Parenteral norovirus vaccine formulations

ABSTRACT

The present invention relates to single dose parental vaccine compositions comprising mixtures of monovalent Norovirus virus-like particles. Methods of conferring protective immunity against Norovirus infections in a human subject by administering such compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the Divisional of application Ser. No. 14/796,614,filed Jul. 10, 2015, which is a Continuation of application Ser. No.13/840,403, filed Mar. 15, 2013, now U.S. Pat. No. 9,801,934, which is aContinuation of PCT/US2012/046222, filed Jul. 11, 2012, which claims thebenefit of Provisional Application 61/506,447, filed on Jul. 11, 2011,the contents of each are herein incorporated by reference in theirentireties.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:LIGO_024_03US_SeqList_ST25, date recorded on Jan. 30, 2018, file size 5kilobytes).

FIELD OF THE INVENTION

The invention is in the field of vaccines, particularly vaccines forNoroviruses. In addition, the invention relates to methods of preparingvaccine compositions and methods of inducing and evaluating protectiveimmune responses against Norovirus in humans.

BACKGROUND OF THE INVENTION

Noroviruses are non-cultivatable human Caliciviruses that have emergedas the single most important cause of epidemic outbreaks of nonbacterialgastroenteritis (Glass et al., 2000; Hardy et al., 1999). The clinicalsignificance of Noroviruses was under-appreciated prior to thedevelopment of sensitive molecular diagnostic assays. The cloning of theprototype genogroup I Norwalk virus (NV) genome and the production ofvirus-like particles (VLPs) from a recombinant Baculovirus expressionsystem led to the development of assays that revealed widespreadNorovirus infections (Jiang et al. 1990; 1992).

Noroviruses are single-stranded, positive sense RNA viruses that containa non-segmented RNA genome. The viral genome encodes three open readingframes, of which the latter two specify the production of the majorcapsid protein and a minor structural protein, respectively (Glass etal. 2000). When expressed at high levels in eukaryotic expressionsystems, the capsid protein of NV, and certain other Noroviruses,self-assembles into VLPs that structurally mimic native Norovirusvirions. When viewed by transmission electron microscopy, the VLPs aremorphologically indistinguishable from infectious virions isolated fromhuman stool samples.

Immune responses to Noroviruses are complex, and the correlates ofprotection are just now being elucidated. Human volunteer studiesperformed with native virus demonstrated that mucosally-derived memoryimmune responses provided short-term protection from infection andsuggested that vaccine-mediated protection is feasible (Lindesmith etal. 2003; Parrino et al. 1977; Wyatt et al., 1974).

Although Norovirus cannot be cultivated in vitro, due to theavailability of VLPs and their ability to be produced in largequantities, considerable progress has been made in defining theantigenic and structural topography of the Norovirus capsid. VLPspreserve the authentic confirmation of the viral capsid protein whilelacking the infectious genetic material. Consequently, VLPs mimic thefunctional interactions of the virus with cellular receptors, therebyeliciting an appropriate host immune response while lacking the abilityto reproduce or cause infection. In conjunction with the NIH, BaylorCollege of Medicine studied the humoral, mucosal and cellular immuneresponses to NV VLPs in human volunteers in an academic,investigator-sponsored Phase I clinical trial. Orally administered VLPswere safe and immunogenic in healthy adults (Ball et al. 1999; Tacket etal. 2003). But, multiple doses of a relatively high amount of VLPs wererequired to observe an immune response. At other academic centers,preclinical experiments in animal models have demonstrated enhancementof immune responses to VLPs when administered intranasally withbacterial exotoxin adjuvants (Guerrero et al. 2001; Nicollier-Jamot etal. 2004; Periwal et al. 2003; Souza et al. (2007) Vaccine, Vol.25(50):8448-59). However, protective immunity against Norovirus inhumans remains elusive because the indicators of a protective immuneresponse in humans have still not been clearly identified(Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307).

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that a singledose of a Norovirus vaccine elicits a rapid, robust protective immuneresponse against Norovirus in humans when administered parenterally.Accordingly, the present invention provides a method of elicitingprotective immunity against Norovirus in a human comprisingadministering parenterally to the human no more than a single dose of avaccine composition, said composition comprising genogroup I and/orgenogroup II Norovirus VLPs, wherein said composition induces at least athree-fold increase in Norovirus-specific serum antibody titer ascompared to the titer in the human prior to administration of thecomposition. In certain embodiments, the increase in Norovirus-specificantibody titer is induced within seven days of administration of thesingle dose of the composition. In some embodiments, the vaccinecomposition is administered to the human via an intravenous,subcutaenous, intradermal, or intramuscular route of administration. Inone embodiment, the vaccine composition is administered to the human byan intramuscular route of administration.

The single dose vaccine compositions can comprise doses of about 5 μg toabout 150 of genogroup I Norovirus VLPs, genogroup II Norovirus VLPs, orboth. In embodiments in which the single dose vaccine compositionscomprise both genogroup I and genogroup II Norovirus VLPs, the dose ofeach VLP can be the same or different. In one embodiment, thecomposition comprises no more than 50 μg of genogroup I Norovirus VLPs.In another embodiment, the composition comprises no more than 25 μg ofgenogroup I Norovirus VLPs. In yet another embodiment, the compositioncomprises no more than 150 μg of genogroup II Norovirus VLPs. In stillanother embodiment, the composition comprises no more than 50 μg ofgenogroup II Norovirus VLPs. The Norovirus VLPs can be monovalent VLPsor multivalent VLPs.

In some aspects of the invention, genogroup I Norovirus VLPs in thevaccine compositions comprise a capsid protein derived from a genogroupI viral strain. In one embodiment, the genogroup I Norovirus VLPscomprise a capsid protein from a genogroup I, genotype 1 Norovirus. Inanother embodiment, the genogroup I Norovirus VLPs comprise a capsidprotein from Norwalk virus. In other aspects of the invention, genogroupII Norovirus VLPs in the vaccine compositions comprise a capsid proteinderived from a genogroup II viral strain. In some embodiments, thegenogroup II Norovirus VLPs comprise a capsid protein from a genogroupII, genotype 4 Norovirus. In certain embodiments, the genogroup IINorovirus VLPs are VLPs generated from expression of a consensussequence of genogroup II Norovirus. In one particular embodiment, thegenogroup II Norovirus VLPs comprise a capsid protein having a sequenceof SEQ ID NO: 1.

In certain embodiments, the vaccine composition further comprises atleast one adjuvant. The adjuvant is preferably not a bacterial exotoxinadjuvant. In one embodiment, the adjuvant is a toll-like receptoragonist, such as monophosphoryl lipid A (MPL), flagellin, or CpG. Inanother embodiment, the adjuvant is aluminum hydroxide (e.g. alum). Incertain embodiments, the vaccine composition comprises two adjuvants,such as MPL and aluminum hydroxide. In some embodiments, the vaccinecomposition may further comprise a buffer, such as L-histidine,imidazole, succinic acid, tris, and citric acid. The vaccine compositioncan be formulated as a dry powder or a liquid. In one embodiment, thevaccine composition is formulated as a liquid (e.g. aqueousformulation).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1B. Results of pan-ELISA assays measuring combined serumIgG, IgA, and IgM levels from human volunteers immunized intramuscularlywith placebo (saline) or a vaccine formulation containing 5, 15, 50, or150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4Norovirus VLP. The geometric mean titer for anti-GI.1 (FIG. 1A) andanti-GII.4 (FIG. 1B) antibodies is shown for each of the dosage levelsat 7 and 21 days after the first immunization and 7 and 28 days afterthe second immunization. Volunteers received immunizations on study days0 and 28.

FIG. 2A-FIG. 2B. Results of pan-ELISA assays measuring combined serumIgG, IgA, and IgM levels from human volunteers immunized intramuscularlywith placebo (saline) or a vaccine formulation containing 5, 15, 50, or150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4Norovirus VLP. The geometric mean fold rise for anti-GI.1 (FIG. 2A) andanti-GII.4 (FIG. 2B) antibodies is shown for each of the dosage levelsat 7 and 21 days after the first immunization and 7 and 28 days afterthe second immunization. Volunteers received immunizations on study days0 and 28.

FIG. 3A-FIG. 3B. Results of pan-ELISA assays measuring combined serumIgG, IgA, and IgM levels from human volunteers immunized intramuscularlywith placebo (saline) or a vaccine formulation containing 5, 15, 50, or150 μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4Norovirus VLP. The percent seroresponse rates (i.e. four-fold increasein antibody titer compared to pre-immunization titers) for anti-GI.1(FIG. 3A) and anti-GII.4 (FIG. 3B) antibodies are shown for each of thedosage levels at 7 and 21 days after the first immunization and 7 and 28days after the second immunization. Volunteers received immunizations onstudy days 0 and 28.

FIG. 4A-FIG. 4B. Results of ELISA assays measuring serum IgA from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The geometric mean titer foranti-GI.1 (FIG. 4A) and anti-GII.4 (FIG. 4B) antibodies is shown foreach of the dosage levels at 7 and 21 days after the first immunizationand 7 and 28 days after the second immunization. Volunteers receivedimmunizations on study days 0 and 28.

FIG. 5A-FIG. 5B. Results of ELISA assays measuring serum IgA from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The geometric mean fold rise foranti-GI.1 (FIG. 5A) and anti-GII.4 (FIG. 5B) antibodies is shown foreach of the dosage levels at 7 and 21 days after the first immunizationand 7 and 28 days after the second immunization. Volunteers receivedimmunizations on study days 0 and 28.

FIG. 6A-FIG. 6B. Results of ELISA assays measuring serum IgA from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The percent seroresponse rates(i.e. four-fold increase in antibody titer compared to pre-immunizationtiters) for anti-GI.1 (FIG. 6A) and anti-GII.4 (FIG. 6B) antibodies areshown for each of the dosage levels at 7 and 21 days after the firstimmunization and 7 and 28 days after the second immunization. Volunteersreceived immunizations on study days 0 and 28.

FIG. 7A-FIG. 7B. Results of ELISA assays measuring serum IgG from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The geometric mean titer foranti-GI.1 (FIG. 7A) and anti-GII.4 (FIG. 7B) antibodies is shown foreach of the dosage levels at 7 and 21 days after the first immunizationand 7 and 28 days after the second immunization. Volunteers receivedimmunizations on study days 0 and 28.

FIG. 8A-FIG. 8B. Results of ELISA assays measuring serum IgG from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The geometric mean fold rise foranti-GI.1 (FIG. 8A) and anti-GII.4 (FIG. 8B) antibodies is shown foreach of the dosage levels at 7 and 21 days after the first immunizationand 7 and 28 days after the second immunization. Volunteers receivedimmunizations on study days 0 and 28.

FIG. 9A-FIG. 9B. Results of ELISA assays measuring serum IgG from humanvolunteers immunized intramuscularly with placebo (saline) or a vaccineformulation containing 5, 15, or 50 μg each of a genogroup I.1 NorovirusVLP and a genogroup II.4 Norovirus VLP. The percent seroresponse rates(i.e. four-fold increase in antibody titer compared to pre-immunizationtiters) for anti-GI.1 (FIG. 9A) and anti-GII.4 (FIG. 9B) antibodies areshown for each of the dosage levels at 7 and 21 days after the firstimmunization and 7 and 28 days after the second immunization. Volunteersreceived immunizations on study days 0 and 28.

FIG. 10. Results of pan-ELISA assays measuring combined serum IgG, IgA,and IgM levels from human volunteers immunized with either a Norovirusintranasal, monovalent vaccine as described in El Kamary et al. (2010) JInfect Dis, Vol. 202(11): 1649-1658 (LV01-103 groups) or a Norovirusintramuscular, bivalent vaccine as described in Example 1 (LV03-104groups) at the indicated time points. Human volunteers received eitherplacebo or two doses of either the intramuscular or intranasal vaccineformulation. The intramuscular, bivalent Norovirus vaccine contained 5μg each of a genogroup I.1 Norovirus VLP and a genogroup II.4 NorovirusVLP. The intranasal, monovalent vaccine contained 100 μg of a genogroupI.1 Norovirus. Volunteers receiving the intranasal vaccine or placebowere challenged with live Norovirus following the second immunization.

FIG. 11A-FIG. 11B. FACS analysis of peripheral blood mononuclear cellsobtained from human volunteers on Day 0 prior to immunization witheither a 5 μg dose of Norovirus intramuscular, bivalent vaccine (FIG.11A) or placebo (FIG. 11B) and Day 7 post-immunization. CD19+ PBMC aremucosally targeted as evidenced of expression of alpha 4/beta7 homingreceptor and chemokine CCR10 receptor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of eliciting a protectiveimmunity to Norovirus infections in a subject. In particular, thepresent invention provides methods of eliciting a protective immunityagainst Norovirus in a human by parenterally administering to the humanno more than a single dose of a vaccine comprising Norovirus VLPs andoptionally at least one adjuvant, wherein the vaccine confers protectionfrom or amelioration of at least one symptom of Norovirus infection. Theinventors have surprisingly discovered that intramuscular administrationof no more than a single dose of a vaccine composition comprisingNorovirus VLPs to humans induces a rapid (i.e. within 7 days ofimmunization) serum seroconversion (i.e. at least a three-fold increasein antigen-specific serum antibody titers above pre-vaccination levels)that is indicative of a protective immune response against Norovirusinfection and illness. The immune responses induced by this single dosevaccine composition plateau at high antibody titers similar to thatobserved with natural infection by administration of live virus in humanchallenge studies. Interestingly, a boost dose of the vaccine is notrequired as the immune response is not increased upon furtheradministration of an additional vaccine dose.

The invention provides a vaccine composition comprising one or moreNorovirus antigens. By “Norovirus,” “Norovirus (NOR),” “norovirus,” andgrammatical equivalents herein, are meant members of the genus Norovirusof the family Caliciviridae. In some embodiments, a Norovirus caninclude a group of related, positive-sense single-stranded RNA,nonenveloped viruses that can be infectious to human or non-humanmammalian species. In some embodiments, a Norovirus can cause acutegastroenteritis in humans. Noroviruses also can be referred to as smallround structured viruses (SRSVs) having a defined surface structure orragged edge when viewed by electron microscopy.

Included within the Noroviruses are at least five genogroups (GI, GII,GIII, GIV, and GV). GI, GII, and GIV Noroviruses are infectious inhumans, while GIII Noroviruses primarily infect bovine species. GV hasrecently been isolated from mice (Zheng et al. (2006) Virology, Vol 346:312-323). Representative of GIII are the Jena and Newbury strains, whilethe Alphatron, Fort Lauderdale, and Saint Cloud strains arerepresentative of GIV. The GI and GII groups may be further segregatedinto genetic clusters or genotypes based on genetic classification (Andoet al. (2000) J. Infectious Diseases, Vol. 181(Supp2):5336-5348; Lindellet al. (2005) J. Clin. Microbiol., Vol. 43(3): 1086-1092). As usedherein, the term genetic clusters is used interchangeably with the termgenotypes. Within genogroup I, there are 8 GI clusters known to date(with prototype virus strain name): GI.1 (Norwalk (NV-USA93)); GI.2(Southhampton (SOV-GBR93)); GI.3 (Desert Shield (DSV-USA93)); GI.4(Cruise Ship virus/Chiba (Chiba-JPN00)); GI.5 (318/Musgrove(Musgrov-GBR00)); GI.6 (Hesse (Hesse-DEU98)); GI.7 (Wnchest-GBR00); andGI.8 (Boxer-USA02). Within genogroup II, there are 19 GII clusters knownto date (with prototype virus strain name): GII.1 (Hawaii(Hawaii-USA94)); GII.2 (Snow Mountain/Melksham (Msham-GBR95)); GII.3(Toronto (Toronto-CAN93)); GII.4 (Bristol/Lordsdale (Bristol-GBR93));GII.5 (290/Hillingdon (Hilingd-GBR00)); GII.6 (269/Seacroft(Seacrof-GBR00)); GII.7 (273/Leeds (Leeds-GBR00)); GII.8 (539/Amsterdam(Amstdam-NLD99)); GII.9 (378 (VABeach-USA01)), GII.10 (Erfurt-DEU01);GII.11 (SW9180JPN01); GII.12 (Wortley-GBR00); GII.13 (Faytvil-USA02);GII.14 (M7-USA03); GII.15 (J23-USA02); GII.16 (Tiffin-USA03); GII.17(CSE1-USA03); GII.18 (QW101/2003/US) and GII.19 (QW170/2003/US).

By “Norovirus” also herein is meant recombinant Norovirus virus-likeparticles (rNOR VLPs). In some embodiments, recombinant expression of atleast the Norovirus capsid protein encoded by ORF2 in cells, e.g., froma baculovirus vector in Sf9 cells, can result in spontaneousself-assembly of the capsid protein into VLPs. In some embodiments,recombinant expression of at least the Norovirus proteins encoded byORF1 and ORF2 in cells, e.g., from a baculovirus vector in 519 cells,can result in spontaneous self-assembly of the capsid protein into VLPs.VLPs are structurally similar to Noroviruses but lack the viral RNAgenome and therefore are not infectious. Accordingly, “Norovirus”includes virions that can be infectious or non-infectious particles,which include defective particles.

Non-limiting examples of Noroviruses include Norovirus genogroup 1strain Hu/NoV/West Chester/2001/USA, GenBank Accession No. AY502016;Chiba virus (CHV, GenBank AB042808); Norovirus genogroup 2 strainHu/NoV/Braddock Heights/1999/USA, GenBank Accession No. AY502015;Norovirus genogroup 2 strain Hu/NoV/Fayette/1999/USA, GenBank AccessionNo. AY502014; Norovirus genogroup 2 strain Hu/NoV/Fairfield/1999/USA,GenBank Accession No. AY502013; Norovirus genogroup 2 strainHu/NoV/Sandusky/1999/USA, GenBank Accession No. AY502012; Norovirusgenogroup 2 strain Hu/NoV/Canton/1999/USA, GenBank Accession No.AY502011; Norovirus genogroup 2 strain Hu/NoV/Tiffin/1999/USA, GenBankAccession No. AY502010; Norovirus genogroup 2 strainHu/NoV/CS-E1/2002/USA, GenBank Accession No. AY50200; Norovirusgenogroup 1 strain Hu/NoV/Wisconsin/2001/USA, GenBank Accession No.AY502008; Norovirus genogroup 1 strain Hu/NoV/CS-841/2001/USA, GenBankAccession No. AY502007; Norovirus genogroup 2 strainHu/NoV/Hiram/2000/USA, GenBank Accession No. AY502006; Norovirusgenogroup 2 strain Hu/NoV/Tontogany/1999/USA, GenBank Accession No.AY502005; Norwalk virus, complete genome, GenBank Accession No.NC.sub.-001959; Norovirus Hu/GI/Otofuke/1979/JP genomic RNA, completegenome, GenBank Accession No. AB187514; NorovirusHu/Hokkaido/133/2003/JP, GenBank Accession No. AB212306; NorovirusSydney 2212, GenBank Accession No. AY588132; Norwalk virus strainSN2000JA, GenBank Accession No. AB190457; Lordsdale virus completegenome, GenBank Accession No. X86557; Norwalk-like virus genomic RNA,Gifu'96, GenBank Accession No. AB045603; Norwalk virus strain Vietnam026, complete genome, GenBank Accession No. AF504671; NorovirusHu/GII.4/2004/N/L, GenBank Accession No. AY883096; NorovirusHu/GII/Hokushin/03/JP, GenBank Accession No. AB195227; NorovirusHu/GII/Kamo/03/JP, GenBank Accession No. AB195228; NorovirusHu/GII/Sinsiro/97/JP, GenBank Accession No. AB195226; NorovirusHu/GII/Ina/02/JP, GenBank Accession No. AB 195225; NorovirusHu/NLV/GII/Neustrelitz260/2000/DE, GenBank Accession No. AY772730;Norovirus Hu/NLV/Dresden174/pUS-NorII/1997/GE, GenBank Accession No.AY741811; Norovirus Hu/NLV/Oxford/B2S16/2002/UK, GenBank Accession No.AY587989; Norovirus Hu/NLV/Oxford/B4S7/2002/UK, GenBank Accession No.AY587987; Norovirus Hu/NLV/Witney/B7S2/2003/UK, GenBank Accession No.AY588030; Norovirus Hu/NLV/Banbury/B9S23/2003/UK, GenBank Accession No.AY588029; Norovirus Hu/NLV/ChippingNorton/2003/UK, GenBank Accession No.AY588028; Norovirus Hu/NLV/Didcot/B9S2/2003/UK, GenBank Accession No.AY588027; Norovirus Hu/NLV/Oxford/B8S5/2002/UK, GenBank Accession No.AY588026; Norovirus Hu/NLV/Oxford/B6S4/2003/UK, GenBank Accession No.AY588025; Norovirus Hu/NLV/Oxford/B6S5/2003/UK, GenBank Accession No.AY588024; Norovirus Hu/NLV/Oxford/B5S23/2003/UK, GenBank Accession No.AY588023; Norovirus Hu/NLV/Oxford/B6S2/2003/UK, GenBank Accession No.AY588022; Norovirus Hu/NLV/Oxford/B6S6/2003/UK, GenBank Accession No.AY588021; Norwalk-like virus isolate Bo/Thirsk10/00/UK, GenBankAccession No. AY126468; Norwalk-like virus isolate Bo/Penrith55/00/UK,GenBank Accession No. AY126476; Norwalk-like virus isolateBo/Aberystwyth24/00/UK, GenBank Accession No. AY126475; Norwalk-likevirus isolate Bo/Dumfries/94/UK, GenBank Accession No. AY126474;Norovirus NLV/IF2036/2003/Iraq, GenBank Accession No. AY675555;Norovirus NLV/IF1998/2003/Iraq, GenBank Accession No. AY675554;Norovirus NLV/BUDS/2002/USA, GenBank Accession No. AY660568; NorovirusNLV/Paris Island/2003/USA, GenBank Accession No. AY652979; Snow Mountainvirus, complete genome, GenBank Accession No. AY134748; Norwalk-likevirus NLV/Fort Lauderdale/560/1998/US, GenBank Accession No. AF414426;Hu/Norovirus/hiroshima/1999/JP(9912-02F), GenBank Accession No.AB044366; Norwalk-like virus strain 11MSU-MW, GenBank Accession No.AY274820; Norwalk-like virus strain B-1SVD, GenBank Accession No.AY274819; Norovirus genogroup 2 strain Hu/NoV/Farmington Hills/2002/USA,GenBank Accession No. AY502023; Norovirus genogroup 2 strainHu/NoV/CS-G4/2002/USA, GenBank Accession No. AY502022; Norovirusgenogroup 2 strain Hu/NoV/CS-G2/2002/USA, GenBank Accession No.AY502021; Norovirus genogroup 2 strain Hu/NoV/CS-G12002/USA, GenBankAccession No. AY502020; Norovirus genogroup 2 strainHu/NoV/Anchorage/2002/USA, GenBank Accession No. AY502019; Norovirusgenogroup 2 strain Hu/NoV/CS-D1/2002/CAN, GenBank Accession No.AY502018; Norovirus genogroup 2 strain Hu/NoV/Germanton/2002/USA,GenBank Accession No. AY502017; Human calicivirusNLV/GII/Langen1061/2002/DE, complete genome, GenBank Accession No.AY485642; Murine norovirus 1 polyprotein, GenBank Accession No.AY228235; Norwalk virus, GenBank Accession No. AB067536; Humancalicivirus NLV/Mex7076/1999, GenBank Accession No. AF542090; Humancalicivirus NLV/Oberhausen 455/01/DE, GenBank Accession No. AF539440;Human calicivirus NLV/Herzberg 385/01/DE, GenBank Accession No.AF539439; Human calicivirus NLV/Boxer/2001/US, GenBank Accession No.AF538679; Norwalk-like virus genomic RNA, complete genome, GenBankAccession No. AB081723; Norwalk-like virus genomic RNA, complete genome,isolate:Saitama U201, GenBank Accession No. AB039782; Norwalk-like virusgenomic RNA, complete genome, isolate:Saitama U18, GenBank Accession No.AB039781; Norwalk-like virus genomic RNA, complete genome,isolate:Saitama U25, GenBank Accession No. AB039780; Norwalk virusstrain:U25G11, GenBank Accession No. AB067543; Norwalk virus strain:U201GII, GenBank Accession No. AB067542; Norwalk-like viruses strain416/97003156/1996/LA, GenBank Accession No. AF080559; Norwalk-likeviruses strain 408/97003012/1996/FL, GenBank Accession No. AF080558;Norwalk-like virus NLV/Burwash Landing/331/1995/US, GenBank AccessionNo. AF414425; Norwalk-like virus NLV/Miami Beach/326/1995/US, GenBankAccession No. AF414424; Norwalk-like virus NLV/White River/290/1994/US,GenBank Accession No. AF414423; Norwalk-like virus NLV/NewOrleans/306/1994/US, GenBank Accession No. AF414422; Norwalk-like virusNLV/Port Canaveral/301/1994/US, GenBank Accession No. AF414421;Norwalk-like virus NLV/Honolulu/314/1994/US, GenBank Accession No.AF414420; Norwalk-like virus NLV/Richmond/283/1994/US, GenBank AccessionNo. AF414419; Norwalk-like virus NLV/Westover/302/1994/US, GenBankAccession No. AF414418; Norwalk-like virus NLV/UK3-17/12700/1992/GB,GenBank Accession No. AF414417; Norwalk-like virus NLV/Miami/81/1986/US,GenBank Accession No. AF414416; Snow Mountain strain, GenBank AccessionNo. U70059; Desert Shield virus DSV395, GenBank Accession No. U04469;Norwalk virus, complete genome, GenBank Accession No. AF093797; Hawaiicalicivirus, GenBank Accession No. U07611; Southampton virus, GenBankAccession No. L07418; Norwalk virus (SRSV-KY-89/89/J), GenBank AccessionNo. L23828; Norwalk virus (SRSV-SMA/76/US), GenBank Accession No.L23831; Camberwell virus, GenBank Accession No. U46500; Humancalicivirus strain Melksham, GenBank Accession No. X81879; Humancalicivirus strain MX, GenBank Accession No. U22498; Minireovirus TV24,GenBank Accession No. U02030; and Norwalk-like virus NLV/Gnedd/273/1994/US, GenBank Accession No. AF414409; sequences of all ofwhich (as entered by the date of filing of this application) are hereinincorporated by reference. Additional Norovirus sequences are disclosedin the following patent publications: WO 2005/030806, WO 2000/79280,JP2002020399, US2003129588, U.S. Pat. No. 6,572,862, WO 1994/05700, andWO 05/032457, all of which are herein incorporated by reference in theirentireties. See also Green et al. (2000) J. Infect. Dis., Vol.181(Suppl. 2):5322-330; Wang et al. (1994) J. Virol., Vol. 68:5982-5990;Chen et al. (2004) J. Virol., Vol. 78: 6469-6479; Chakravarty et al.(2005) J. Virol., Vol. 79: 554-568; Hansman et al. (2006) J. Gen.Virol., Vol. 87:909-919; Bull et al. (2006) J. Clin. Micro., Vol.44(2):327-333; Siebenga, et al. (2007) J. Virol., Vol. 81(18):9932-9941,and Fankhauser et al. (1998) J. Infect. Dis., Vol. 178:1571-1578; forsequence comparisons and a discussion of genetic diversity andphylogenetic analysis of Noroviruses. The nucleic acid and correspondingamino acid sequences of each are all incorporated by reference in theirentirety. In some embodiments, a cryptogram can be used foridentification purposes and is organized: host species from which thevirus was isolated/genus abbreviation/species abbreviation/strainname/year of occurrence/country of origin. (Green et al., HumanCaliciviruses, in Fields Virology Vol. 1 841-874 (Knipe and Howley,editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)).Genogroup II, genotype 4 (GII.4) viral strains (e.g., Houston, Minerva(also known as Den Haag), and Laurens (also known as Yerseke) strains)are preferred in some embodiments. As new strains are identified andtheir genetic sequences are made available, one skilled in the art wouldbe able to employ VLPs using these contemporary strains in thecompositions and methods of the present invention using ordinary skill.Thus, the present invention contemplates VLPs made from such strains assuitable antigens for use in the compositions and methods describedherein.

The Norovirus antigen may be in the form of peptides, proteins, orvirus-like particles (VLPs). In a preferred embodiment, the Norovirusantigen comprises VLPs. As used herein, “virus-like particle(s) or VLPs”refer to a virus-like particle(s), fragment(s), aggregates, orportion(s) thereof produced from the capsid protein coding sequence ofNorovirus and comprising antigenic characteristic(s) similar to those ofinfectious Norovirus particles. Norovirus antigens may also be in theform of capsid monomers, capsid multimers, protein or peptide fragmentsof VLPs, or aggregates or mixtures thereof. The Norovirus antigenicproteins or peptides may also be in a denatured form, produced usingmethods known in the art.

The VLPs of the present invention can be formed from either the fulllength Norovirus capsid protein such as VP1 and/or VP2 proteins orcertain VP1 or VP2 derivatives using standard methods in the art.Alternatively, the capsid protein used to form the VLP is a truncatedcapsid protein. In some embodiments, for example, at least one of theVLPs comprises a truncated VP1 protein. In other embodiments, all theVLPs comprise truncated VP1 proteins. The truncation may be an N- orC-terminal truncation. Truncated capsid proteins are suitably functionalcapsid protein derivatives. Functional capsid protein derivatives arecapable of raising an immune response (if necessary, when suitablyadjuvanted) in the same way as the immune response is raised by a VLPconsisting of the full length capsid protein.

VLPs may contain major VP1 proteins and/or minor VP2 proteins. In someembodiments, each VLP contains VP1 and/or VP2 protein from only oneNorovirus genogroup giving rise to a monovalent VLP. As used herein, theterm “monovalent” means the antigenic proteins are derived from a singleNorovirus genogroup. For example, the VLPs contain VP1 and/or VP2 from avirus strain of genogroup I (e.g., VP1 and VP2 from Norwalk virus).Preferably the VLP is comprised of predominantly VP1 proteins. In oneembodiment of the invention, the antigen is a mixture of monovalent VLPswherein the composition includes VLPs comprised of VP1 and VP2 from asingle Norovirus genogroup mixed with VLPs comprised of VP1 and VP2 froma different Norovirus genogroup (e.g. Norwalk virus and Houston virus)taken from multiple viral strains. Purely by way of example thecomposition can contain monovalent VLPs from one or more strains ofNorovirus genogroup I together with monovalent VLPs from one or morestrains of Norovirus genogroup II. Strains may be selected based ontheir predominance of circulation at a given time. In certainembodiments, the Norovirus VLP mixture is composed of GI.1 and GII.4viral strains. More preferably, the Norovirus VLP mixture is composed ofthe strains of Norwalk and a consensus capsid sequence derived fromgenogroup II Noroviruses. Consensus capsid sequences derived fromcirculating Norovirus sequences and VLPs made with such sequences aredescribed in WO 2010/017542, which is herein incorporated by referencein its entirety. For instance, in one embodiment, a consensus capsidsequence derived from genogroup II, genotype 4 (GII.4) viral strainscomprises a sequence of SEQ ID NO: 1. Thus, in some embodiments, thevaccine composition comprises a mixture of monovalent VLPs, wherein onemonovalent VLP comprises a capsid protein from a genogroup I Norovirus(e.g. Norwalk) and the other monovalent VLP comprises a consensus capsidprotein comprising a sequence of SEQ ID NO: 1.

(SEQ ID NO: 1)M K M A S S D A N P S D G S T A N L V P E V N N E V M A L E P VV G A A I A A P V A G Q Q N V I D P W I R N N F V Q A P G G E FT V S P R N A P G E I L W S A P L G P D L N P Y L S H L A R M YN G Y A G G F E V Q V I L A G N A F T A G K I I F A A V P P N FP T E G L S P S Q V T M F P H I I V D V R Q L E P V L I P L P DV R N N F Y H Y N Q S N D P T I K L I A M L Y T P L R A N N A GD D V F T V S C R V L T R P S P D F D F I F L V P P T V E S R TK P F T V P I L T V E E M T N S R F P I P L E K L F T G P S G AF V V Q P Q N G R C T T D G V L L G T T Q L S P V N I C T F R GD V T H I A G T Q E Y T M N L A S Q N W N N Y D P T E E I P A PL G T P D F V G K I Q G V L T Q T T R G D G S T R G H K A T V ST G S V H F T P K L G S V Q F S T D T S N D F E T G Q N T K F TP V G V V Q D G S T T H Q N E P Q Q W V L P D Y S G R D S H N VH L A P A V A P T F P G E Q L L F F R S T M P G C S G Y P N M NL D C L L P Q E W V Q H F Y Q E A A P A Q S D V A L L R F V N PD T G R V L F E C K L H K S G Y V T V A H T G Q H D L V I P P NG Y F R F D S W V N Q F Y T L A P M G N G T G R R R A L

However, in an alternative embodiment of the invention, the VLPs may bemultivalent VLPs that comprise, for example, VP1 and/or VP2 proteinsfrom one Norovirus genogroup intermixed with VP1 and/or VP2 proteinsfrom a second Norovirus genogroup, wherein the different VP1 and VP2proteins are not chimeric VP1 and VP2 proteins, but associate togetherwithin the same capsid structure to form immunogenic VLPs. As usedherein, the term “multivalent” means that the antigenic proteins arederived from two or more Norovirus genogroups or strains. MultivalentVLPs may contain VLP antigens taken from two or more viral strains.Purely by way of example the composition can contain multivalent VLPscomprised of capsid monomers or multimers from one or more strains ofNorovirus genogroup I (e.g. Norwalk virus) together with capsid monomersor multimers from one or more strains of Norovirus genogroup II (e.g.Houston virus). Preferably, the multivalent VLPs contain capsid proteinsfrom the strains of Norwalk and Houston Noroviruses, or otherpredominantly circulating strains at a given time.

The combination of monovalent or multivalent VLPs within the compositionpreferably would not reduce the immunogenicity of each VLP type. Inparticular it is preferred that there is no interference betweenNorovirus VLPs in the combination of the invention, such that thecombined VLP composition of the invention is able to elicit immunityagainst infection by each Norovirus genotype represented in the vaccine.Suitably the immune response against a given VLP type in the combinationis at least 50% of the immune response of that same VLP type whenmeasured individually, preferably 100% or substantially 100%. The immuneresponse may suitably be measured, for example, by antibody responses,as illustrated in the examples herein.

As used herein, “genogroup I Norovirus VLPs” refer to either monovalentor multivalent VLPs that comprise a capsid protein derived from one ormore genogroup I Norovirus strains. In some embodiments, genogroup INorovirus VLPs comprise a full length capsid protein from a genogroup INorovirus (e.g. Norwalk virus). In other embodiments, genogroup INorovirus VLPs comprise a consensus capsid protein derived from variousgenogroup I strains. The genogroup I strains from which the consensuscapsid sequence is derived can be within the same genotype or geneticcluster or from different genotypes or genetic clusters. Similarly, asused herein, “genogroup II Norovirus VLPs” refer to either monovalent ormultivalent VLPs that comprise a capsid protein derived from one or moregenogroup II Norovirus strains. In some embodiments, genogroup IINorovirus VLPs comprise a full length capsid protein from a genogroup IINorovirus (e.g. Laurens or Minerva virus). In other embodiments,genogroup II Norovirus VLPs comprise a consensus capsid protein derivedfrom various genogroup II strains. The genogroup II strains from whichthe consensus capsid sequence is derived can be within the same genotypeor genetic cluster or from different genotypes or genetic clusters. Inone embodiment, the genogroup II Norovirus VLPs comprise a capsidconsensus sequence of genogroup II, genotype 4 (GII.4) Norovirus. Thus,in some embodiments, the genogroup II Norovirus VLPs comprise a capsidsequence of SEQ ID NO: 1.

Multivalent VLPs may be produced by separate expression of theindividual capsid proteins followed by combination to form VLPs.Alternatively multiple capsid proteins may be expressed within the samecell, from one or more DNA constructs. For example, multiple DNAconstructs may be transformed or transfected into host cells, eachvector encoding a different capsid protein. Alternatively a singlevector having multiple capsid genes, controlled by a shared promoter ormultiple individual promoters, may be used. IRES elements may also beincorporated into the vector, where appropriate. Using such expressionstrategies, the co-expressed capsid proteins may be co-purified forsubsequent VLP formation, or may spontaneously form multivalent VLPswhich can then be purified.

A preferred process for multivalent VLP production comprises preparationof VLP capsid proteins or derivatives, such as VP1 proteins, fromdifferent Norovirus genotypes, mixing the proteins, and assembly of theproteins to produce multivalent VLPs. The VP1 proteins may be in theform of a crude extract, be partially purified or purified prior tomixing. Assembled monovalent VLPs of different genogroups may bedisassembled, mixed together and reassembled into multivalent VLPs.Preferably the proteins or VLPs are at least partially purified beforebeing combined. Optionally, further purification of the multivalent VLPsmay be carried out after assembly.

Suitably the VLPs of the invention are made by disassembly andreassembly of VLPs, to provide homogenous and pure VLPs. In oneembodiment multivalent VLPs may be made by disassembly of two or moreVLPs, followed by combination of the disassembled VLP components at anysuitable point prior to reassembly. This approach is suitable when VLPsspontaneously form from expressed VP1 protein, as occurs for example, insome yeast strains. Where the expression of the VP1 protein does notlead to spontaneous VLP formation, preparations of VP1 proteins orcapsomers may be combined before assembly into VLPs.

Where multivalent VLPs are used, preferably the components of the VLPsare mixed in the proportions in which they are desired in the finalmixed VLP. For example, a mixture of the same amount of a partiallypurified VP1 protein from Norwalk and Houston viruses (or otherNorovirus strains) provides a multivalent VLP with approximately equalamounts of each protein.

Compositions comprising multivalent VLPs may be stabilized by solutionsknown in the art, such as those of WO 98/44944, WO 00/45841,incorporated herein by reference.

Compositions of the invention may comprise other proteins or proteinfragments in addition to Norovirus VP1 and VP2 proteins or derivatives.Other proteins or peptides may also be co-administered with thecomposition of the invention. Optionally the composition may also beformulated or co-administered with non-Norovirus antigens. Suitablythese antigens can provide protection against other diseases.

The VP1 protein or functional protein derivative is suitably able toform a VLP, and VLP formation can be assessed by standard techniquessuch as, for example, size exclusion chromatography, electron microscopyand dynamic laser light scattering.

The antigenic molecules of the present invention can be prepared byisolation and purification from the organisms in which they occurnaturally, or they may be prepared by recombinant techniques. Preferablythe Norovirus VLP antigens are prepared from insect cells such as 519 orH5 cells, although any suitable cells such as E. coli or yeast cells,for example, S. cerevisiae, S. pombe, Pichia pastori or other Pichiaexpression systems, mammalian cell expression such as CHO or HEK systemsmay also be used. When prepared by a recombinant method or by synthesis,one or more insertions, deletions, inversions or substitutions of theamino acids constituting the peptide may be made. Each of theaforementioned antigens is preferably used in the substantially purestate.

The procedures of production of norovirus VLPs in insect cell culturehave been previously disclosed in U.S. Pat. No. 6,942,865, which isincorporated herein by reference in its entirety. Briefly, a cDNA fromthe 3′ end of the genome containing the viral capsid gene (ORF2) and aminor structural gene (ORF3) is cloned. The recombinant baculovirusescarrying the viral capsid genes is constructed from the cloned cDNAs.Norovirus VLPs are produced in 519 or H5 insect cell cultures.

In some embodiments, the vaccine composition comprises one or moreadjuvants in combination with the Norovirus antigen. Most adjuvantscontain a substance designed to protect the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as Bordatella pertussis or Mycobacteriumtuberculosis derived proteins. Suitable adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and CompleteAdjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merckand Company, Inc., Rahway, N.J.); aluminum salts such as aluminumhydroxide gel (alum) or aluminum phosphate; salts of calcium, iron orzinc; an insoluble suspension of acylated tyrosine acylated sugars;cationically or anionically derivatized polysaccharides;polyphosphazenes; biodegradable microspheres; and Quil A.

Suitable adjuvants also include, but are not limited to, toll-likereceptor (TLR) agonists, particularly toll-like receptor type 4 (TLR-4)agonists (e.g., monophosphoryl lipid A (MPL), synthetic lipid A, lipid Amimetics or analogs), aluminum salts, cytokines, saponins, muramyldipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) ofgram-negative bacteria, polyphosphazenes, emulsions, virosomes,cochleates, poly(lactide-co-glycolides) (PLG) microparticles, poloxamerparticles, microparticles, liposomes, oil-in-water emulsions, MF59, andsqualene. In some embodiments, the adjuvants are not bacterially-derivedexotoxins. Preferred adjuvants include adjuvants which stimulate a Th1type response such as 3DMPL or QS21.

Monophosphoryl Lipid A (MPL), a non-toxic derivative of lipid A fromSalmonella, is a potent TLR-4 agonist that has been developed as avaccine adjuvant (Evans et al. 2003). In pre-clinical murine studiesintranasal MPL has been shown to enhance secretory, as well as systemic,humoral responses (Baldridge et al. 2000; Yang et al. 2002). It has alsobeen proven to be safe and effective as a vaccine adjuvant in clinicalstudies of greater than 120,000 patients (Baldrick et al., 2002;Baldridge et al. 2004). MPL stimulates the induction of innate immunitythrough the TLR-4 receptor and is thus capable of eliciting nonspecificimmune responses against a wide range of infectious pathogens, includingboth gram negative and gram positive bacteria, viruses, and parasites(Baldridge et al. 2004; Persing et al. 2002). Inclusion of MPL invaccine formulations should provide rapid induction of innate responses,eliciting nonspecific immune responses from viral challenge whileenhancing the specific responses generated by the antigenic componentsof the vaccine.

In one embodiment, the present invention provides a compositioncomprising monophosphoryl lipid A (MPL) or 3 De-O-acylatedmonophosphoryl lipid A (3D-MPL) as an enhancer of adaptive and innateimmunity. Chemically 3D-MPL is a mixture of 3 De-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred formof 3 De-O-acylated monophosphoryl lipid A is disclosed in EuropeanPatent 0 689 454 B1 (SmithKline Beecham Biologicals SA), which isincorporated herein by reference. In another embodiment, the presentinvention provides a composition comprising synthetic lipid A, lipid Amimetics or analogs, such as BioMira's PET Lipid A, or syntheticderivatives designed to function like TLR-4 agonists.

In certain embodiments, the vaccine composition comprises two adjuvants.A combination of adjuvants may be selected from those described above.In one particular embodiment, the two adjuvants are MPL and aluminumhydroxide (e.g., alum). In another particular embodiment, the twoadjuvants are MPL and oil.

The term “effective adjuvant amount” or “effective amount of adjuvant”will be well understood by those skilled in the art, and includes anamount of one or more adjuvants which is capable of stimulating theimmune response to an administered antigen, i.e., an amount thatincreases the immune response of an administered antigen composition, asmeasured in terms of the IgA levels in the nasal washings, serum IgG orIgM levels, or B and T-Cell proliferation. Suitably effective increasesin immunoglobulin levels include by more than 5%, preferably by morethan 25%, and in particular by more than 50%, as compared to the sameantigen composition without any adjuvant.

In one embodiment, the present invention provides a vaccine compositionformulated for parenteral administration, wherein the compositionincludes at least two types of Norovirus VLPs in combination withaluminum hydroxide and a buffer. The buffer can be selected from thegroup consisting of L-histidine, imidazole, succinic acid, tris, citricacid, bis-tris, pipes, mes, hepes, glycine amide, and tricine. In oneembodiment, the buffer is L-histidine or imidazole. Preferably, thebuffer is present in a concentration from about 15 mM to about 50 mM,more preferably from about 18 mM to about 40 mM, or most preferablyabout 20 mM to about 25 mM. In some embodiments, the pH of the antigenicor vaccine composition is from about 6.0 to about 7.0, or from about 6.2to about 6.8, or about 6.5. The vaccine composition can be an aqueousformulation. In some embodiments, the vaccine composition is alyophilized powder and reconstituted to an aqueous formulation.

In certain embodiments, the vaccine composition further comprises atleast one adjuvant in addition to the two or more types of NorovirusVLPs, aluminum hydroxide, and a buffer. For instance, the adjuvant canbe a toll-like receptor agonist, such as MPL, flagellin, CpG oligos,synthetic lipid A or lipid A mimetics or analogs. In one particularembodiment, the adjuvant is MPL.

The Norovirus VLPs included in the vaccine compositions of the inventioncan be any of the VLPs described herein. In one embodiment, the twotypes of Norovirus VLPs each comprise a capsid protein from differentgenogroups (e.g., genogroup I and genogroup II). For instance, one typeof Norovirus VLP comprises a capsid protein derived from a genogroup INorovirus and the other type of Norovirus VLP comprises a capsid proteinderived from a genogroup II Norovirus. In one embodiment, one type ofNorovirus VLP comprises a capsid protein from Norwalk virus and theother type of Norovirus VLP comprises a consensus capsid protein derivedfrom genogroup II, genotype 4 Noroviruses (e.g., a capsid proteincomprising a sequence of SEQ ID NO: 1). The vaccine composition cancomprise about 5 μg to about 200 μg of each Norovirus VLP, morepreferably about 15 μg to about 50 μg of each Norovirus VLP. In someembodiments, the dose of one type of Norovirus VLP is different than thedose of the other type of Norovirus VLP. For instance, in certainembodiments, the vaccine composition comprises about 5 μg to about 15 μgof a genogroup I VLP and about 15 μg to about 50 μg of a genogroup IIVLP. In other embodiments, the vaccine composition comprises about 15 μgto about 50 μg of a genogroup I VLP and about 50 μg to about 150 μg of agenogroup II VLP.

In some embodiments, the vaccine compositions further comprise apharmaceutically acceptable salt, including, but not limited to, sodiumchloride, potassium chloride, sodium sulfate, ammonium sulfate, andsodium citrate. In one embodiment, the pharmaceutically acceptable saltis sodium chloride. The concentration of the pharmaceutically acceptablesalt can be from about 10 mM to about 200 mM, with preferredconcentrations in the range of from about 100 mM to about 150 mM.Preferably, the vaccine compositions of the invention contain less than2 mM of free phosphate. In some embodiments, the vaccine compositionscomprise less than 1 mM of free phosphate. The vaccine compositions mayalso further comprise other pharmaceutically acceptable excipients, suchas sugars (e.g., sucrose, trehalose, mannitol) and surfactants.

As discussed herein, the compositions of the invention can be formulatedfor administration as vaccines formulations. As used herein, the term“vaccine” refers to a formulation which contains Norovirus VLPs or otherNorovirus antigens of the present invention as described above, which isin a form that is capable of being administered to a vertebrate,particularly a human, and which induces a protective immune responsesufficient to induce immunity to prevent and/or ameliorate a Norovirusinfection or Norovirus-induced illness and/or to reduce at least onesymptom of a Norovirus infection or illness.

As used herein, the term “immune response” refers to both the humoralimmune response and the cell-mediated immune response. The humoralimmune response involves the stimulation of the production of antibodiesby B lymphocytes that, for example, neutralize infectious agents, blockinfectious agents from entering cells, block replication of saidinfectious agents, and/or protect host cells from infection anddestruction. The cell-mediated immune response refers to an immuneresponse that is mediated by T-lymphocytes and/or other cells, such asmacrophages, against an infectious agent, exhibited by a vertebrate(e.g., a human), that prevents or ameliorates infection or reduces atleast one symptom thereof. In particular, “protective immunity” or“protective immune response” refers to immunity or eliciting an immuneresponse against an infectious agent, which is exhibited by a vertebrate(e.g., a human), that prevents or ameliorates an infection or reduces atleast one symptom thereof. Specifically, induction of a protectiveimmune response from administration of the vaccine is evident byelimination or reduction of the presence of one or more symptoms ofacute gastroenteritis or a reduction in the duration or severity of suchsymptoms. Clinical symptoms of gastroenteritis from Norovirus includenausea, diarrhea, loose stool, vomiting, fever, and general malaise. Aprotective immune response that reduces or eliminates disease symptomswill reduce or stop the spread of a Norovirus outbreak in a population.Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). The compositions of the present invention can beformulated, for example, for delivery to one or more of the oral,gastro-intestinal, and respiratory (e.g. nasal) mucosa. The compositionsof the present invention can be formulated, for example, for delivery byinjection, such as parenteral injection (e.g., intravenous,subcutaneous, intradermal, or intramuscular injection).

Where the composition is intended for delivery to the respiratory (e.g.nasal) mucosa, typically it is formulated as an aqueous solution foradministration as an aerosol or nasal drops, or alternatively, as a drypowder, e.g. for rapid deposition within the nasal passage. Compositionsfor administration as nasal drops may contain one or more excipients ofthe type usually included in such compositions, for examplepreservatives, viscosity adjusting agents, tonicity adjusting agents,buffering agents, and the like. Viscosity agents can be microcrystallinecellulose, chitosan, starches, polysaccharides, and the like.Compositions for administration as dry powder may also contain one ormore excipients usually included in such compositions, for example,mucoadhesive agents, bulking agents, and agents to deliver appropriatepowder flow and size characteristics. Bulking and powder flow and sizeagents may include mannitol, sucrose, trehalose, and xylitol.

Where the composition is intended for parenteral injection, such asintravenous (i.v.), subcutaneous (s.c.), intradermal, or intramuscular(i.m.) injection, it is typically formulated as a liquid suspension(i.e. aqueous formulation) comprised of at least one type of NorovirusVLP and optionally at least one adjuvant. In one embodiment, theadjuvant may be MPL. In another embodiment, liquid vaccine formulatedfor parenteral administration may have more than one adjuvant. In apreferred embodiment, a parenterally-formulated (e.g., i.m., i.v., ors.c.-formulated) liquid vaccine comprises Norovirus genogroup I and/orgenogroup II VLPs with aluminum hydroxide (e.g. alum) and monophosphoryllipid A (MPL) as adjuvants. In one embodiment, a liquid formulation forparenteral administration comprises Norovirus genogroup antigen(s), suchas one or more types of Norovirus VLPs as described herein, MPL,aluminum hydroxide, and a buffer. In another embodiment, a liquidformulation for parenteral administration comprises Norovirus genogroupantigen(s), MPL, oil, and a buffer. In certain embodiments, the bufferin the parenteral vaccine formulations is L-histidine or imidazole.Parenteral administration of liquid vaccines can be by needle andsyringe, as is well known in the art.

In certain embodiments, a vaccine composition of the invention foreliciting a protective immune response against Norovirus in humanscomprises genogroup I and/or genogroup II Norovirus VLPs at a dose of nomore than 150 μg. For instance, in some embodiments, the vaccinecomposition comprises no more than 150 μg, no more than 100 μg, no morethan 50 μg, no more than 25 μg, no more than 15 μg, or no more than 10μg of genogroup I Norovirus VLPs. In other embodiments, the vaccinecomposition comprises no more than 150 μg, no more than 100 μg, no morethan 50 μg, no more than 25 μg, no more than 15 μg, or no more than 10μg of genogroup II Norovirus VLPs. In certain embodiments, the vaccinecomposition comprises no more than 150 μg of each genogroup I andgenogroup II Norovirus VLPs. In such embodiments, the dose of genogroupI Norovirus VLPs and genogroup II VLPs can be the same or different. Forinstance, in one embodiment, the vaccine composition may comprise nomore than 50 μg of genogroup I Norovirus VLPs and no more than 150 μg ofgenogroup II Norovirus VLPs. In another embodiment, the vaccinecomposition may comprise no more than 25 μg of genogroup I NorovirusVLPs and no more than 50 μg of genogroup II Norovirus VLPs. In otherembodiments, the vaccine composition may comprise no more than 15 μg ofgenogroup I Norovirus VLPs and no more than 50 μg of genogroup IINorovirus VLPs. In still other embodiments, the vaccine composition maycomprise no more than 25 μg of genogroup I Norovirus VLPs and no morethan 150 μg of genogroup II Norovirus VLPs.

The genogroup I and genogroup II Norovirus VLPs can be derived from anyof the Norovirus strains described herein. In one embodiment, thegenogroup I Norovirus VLPs are genogroup I, genotype 1 (GI.1) VLPs (i.e.comprise a capsid protein from a GI.1 Norovirus). In another embodiment,the genogroup I Norovirus VLPs are Norwalk VLPs. In another embodiment,the genogroup II Norovirus VLPs are genogroup II, genotype 4 (GII.4)VLPs. In still another embodiment, the genogroup II Norovirus VLPs areVLPs generated from expression of a consensus sequence of genogroup IINorovirus. In a particular embodiment, the genogroup II Norovirus VLPscomprise a capsid protein having a sequence of SEQ ID NO: 1.

The vaccine compositions hereinbefore described may be lyophilized andstored anhydrous until they are ready to be used, at which point theyare reconstituted with diluent. Alternatively, different components ofthe composition may be stored separately in a kit (any or all componentsbeing lyophilized). The components may remain in lyophilized form fordry formulation or be reconstituted for liquid formulations, and eithermixed prior to use or administered separately to the patient. In someembodiments, the vaccine compositions are stored in kits in liquidformulations and may be accompanied by delivery devices, such assyringes equipped with needles. In other embodiments, the liquid vaccinecompositions may be stored within the delivery devices in a kit. Forexample, a kit may comprise pre-filled syringes, autoinjectors, orinjection pen devices containing a liquid formulation of a vaccinecomposition described herein.

The lyophilization of vaccines is well known in the art. Typically theliquid antigen is freeze dried in the presence of agents to protect theantigen during the lyophilization process and to yield a cake withdesirable powder characteristics. Sugars such as sucrose, mannitol,trehalose, or lactose (present at an initial concentration of 10-200mg/mL) are commonly used for cryoprotection of protein antigens and toyield lyophilized cake with desirable powder characteristics.Lyophilizing the compositions theoretically results in a more stablecomposition.

The amount of antigen in each vaccine composition is selected as anamount which induces a robust immune response without significant,adverse side effects. Such amount will vary depending upon whichspecific antigen(s) is employed, route of administration, and adjuvantsused. In general, the dose administered to a patient, in the context ofthe present invention should be sufficient to effect a protective immuneresponse in the patient over time, or to induce the production ofantigen-specific antibodies. Thus, the composition is administered to apatient in an amount sufficient to elicit an immune response to thespecific antigens and/or to prevent, alleviate, reduce, or cure symptomsand/or complications from the disease or infection, and thus reduce orstop the spread of a Norovirus outbreak in a population. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.”

The vaccine compositions of the present invention may be administeredvia a non-mucosal or mucosal route. These administrations may include invivo administration via parenteral injection (e.g. intravenous,subcutaneous, intradermal, and intramuscular) or other traditionaldirect routes, such as buccal/sublingual, rectal, oral, nasal, topical(such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial,intraperitoneal, intraocular, or intranasal routes or directly into aspecific tissue. Other suitable routes of administration includetranscutaneous, subdermal, and via suppository. In one embodiment, thevaccine is administered by a parenteral route of administration, such asintravenous, subcutaneous, intradermal, or intramuscular. In certainembodiments, the vaccine is administered by an intramuscular route ofadministration. Administration may be accomplished simply by directadministration using a needle, catheter or related device (e.g.pre-filled syringes or autoinjectors), at a single time point or atmultiple time points. Other parenteral formulations may be deliveredsubcutaneously or intradermally by microinjection or skin patch deliverymethods.

The present invention provides methods for eliciting protective immunityagainst Norovirus in a subject comprising parenterally administering tothe subject no more than a single dose of a vaccine composition of theinvention, wherein said vaccine comprises genogroup I and/or genogroupII Norovirus VLPs as described herein and optionally at least oneadjuvant. In such embodiments, the single dose vaccine compositioninduces at least a three-fold increase in Norovirus-specific serumantibody titer as compared to the titer in the human prior toadministration of the composition. In some embodiments, the single dosevaccine composition induces at least a six-fold increase inNorovirus-specific serum antibody titer as compared to the titer in thehuman prior to administration of the composition. In other embodiments,the single dose vaccine composition induces a Norovirus-specific serumantibody titer comparable to the antibody titer induced by exposure tolive Norovirus in a natural infection—i.e., a greater than ten-foldincrease in Norovirus-specific serum antibody as compared to the titerin the human prior to administration of the composition. In certainembodiments, the single dose vaccine composition induces the increase inNorovirus-specific serum antibody titer within seven days ofadministration of the composition. Preferably, the single dose vaccinecomposition is administered by an intravenous, subcutaneous, orintramuscular route of administration. In a certain embodiment, thesingle dose vaccine composition is administered intramuscularly to thehuman.

As described herein, in some embodiments, the single dose vaccinecompositions suitable for use in the method comprise no more than 150 μgeach of genogroup I and/or genogroup II Noroviruses. For instance, insome embodiments, the vaccine composition comprises no more than 150 μs,no more than 100 μs, no more than 50 μs, no more than 25 μs, no morethan 15 μs, or no more than 10 μg of genogroup I Norovirus VLPs. Inother embodiments, the vaccine composition comprises no more than 150μs, no more than 100 μs, no more than 50 μs, no more than 25 μs, no morethan 15 μg, or no more than 10 μg of genogroup II Norovirus VLPs. Incertain embodiments, the vaccine composition comprises no more than 50μg of each genogroup I and genogroup II Norovirus VLPs. In embodimentsin which the single dose vaccine composition comprises both genogroup Iand genogroup II Norovirus VLPs, the dose of genogroup I Norovirus VLPsand genogroup II VLPs can be the same or different. For instance, in oneembodiment, the vaccine composition may comprise no more than 50 μg ofgenogroup I Norovirus VLPs and no more than 150 μg of genogroup IINorovirus VLPs. In another embodiment, the vaccine composition maycomprise no more than 25 μg of genogroup I Norovirus VLPs and no morethan 50 μg of genogroup II Norovirus VLPs. In other embodiments, thevaccine composition may comprise no more than 15 μg of genogroup INorovirus VLPs and no more than 50 μg of genogroup II Norovirus VLPs. Instill other embodiments, the vaccine composition may comprise no morethan 25 μg of genogroup I Norovirus VLPs and no more than 150 μg ofgenogroup II Norovirus VLPs.

In one embodiment of the method, the subject is a human and the vaccineconfers protection from one or more symptoms of Norovirus infection.Although others have reported methods of inducing an immune responsewith Norovirus antigens (see U.S. Patent Application Publication No. US2007/0207526), the indicators of a protective immune response againstNorovirus in humans have still not been clearly identified(Herbst-Kralovetz et al. (2010) Expert Rev. Vaccines 9(3), 299-307).Unlike several vaccines currently licensed in the U.S. whereeffectiveness of the vaccine correlates with serum antibodies, studieshave shown that markers of an immune response, such as increased titersof serum IgG antibodies against Norwalk virus, are not associated withprotective immunity in humans (Johnson et al. (1990) J. InfectiousDiseases 161: 18-21). Moreover, another study examining Norwalk viralchallenge in humans indicated that susceptibility to Norwalk infectionwas multifactorial and included factors such as secretor status andmemory mucosal immune response (Lindesmith et al. (2003) Nature Medicine9: 548-553).

Because Norovirus is not able to be cultured in vitro, no viralneutralization assays are currently available. A functional assay whichserves as a substitute for the neutralization assay is thehemagglutination inhibition (HAI) assay (see Example 1). HAI measuresthe ability of Norovirus vaccine-induced antibodies to inhibit theagglutination of antigen-coated red blood cells by Norovirus VLPsbecause Norovirus VLPs bind to red blood cell antigens (e.g. histo-bloodgroup antigens). This assay is also known as a carbohydrate blockingassay, as it is indicative of the functional ability of antibodies toblock binding of the virus or VLPs to blood group antigen carbohydrateson a red blood cell. In this assay, a fixed amount of Norovirus VLPs ismixed with a fixed amount of red blood cells and serum from immunizedsubjects. If the serum sample contains functional antibodies, theantibodies will bind to the VLPs, thereby inhibiting the agglutinationof the red blood cells. As used herein, “functional antibodies” refer toantibodies that are capable of inhibiting the interaction betweenNorovirus particles and red blood cell antigens. In other words,functional antibody titer is equivalent to histo-blood group antigen(HBGA) or carbohydrate blocking antibody titer. The serum titer ofNorovirus-specific functional antibodies can be measured by the HAIassay described above. The serum titer of Norovirus-specific functionalantibodies can also be measured using an ELISA-based assay in which acarbohydrate H antigen is bound to microtiter wells and Norovirus VLPbinding to H antigen is detected in the presence of serum (see Example 1and Reeck et al. (2010) J Infect Dis, Vol. 202(8):1212-1218). Anincrease in the level of Norovirus-specific functional antibodies can bean indicator of a protective immune response. Thus, in one embodiment,the administration of the vaccine elicits a protective immunitycomprising an increase in the serum titer of Norovirus-specificfunctional antibodies as compared to the serum titer in a human notreceiving the vaccine. The serum titer of Norovirus-specific functionalantibodies indicative of a protective immune response is preferably ageometric mean titer greater than 40, 50, 75, 100, 125, 150, 175, 200 asmeasured by the HAI assay or blocking titer (BT)₅₀ (50% inhibition of Hantigen binding by Norovirus VLPs) geometric mean titer of greater than100, 150, 200, 250, 300, 350, 400, 450, or 500 as measured by the Hantigen binding assay. In one embodiment, the serum titer ofNorovirus-specific functional antibodies is a geometric mean titergreater than 40 as measured by the HAI assay. In another embodiment, theserum titer of Norovirus-specific functional antibodies is a geometricmean titer greater than 100 as measured by the HAI assay. In anotherembodiment, the serum titer of Norovirus-specific functional antibodiesis a BT₅₀ geometric mean titer greater than 100 as measured by the Hantigen binding assay. In still another embodiment, the serum titer ofNorovirus-specific functional antibodies is a BT₅₀ geometric mean titergreater than 200 as measured by the H antigen binding assay.

In a further aspect, the administration of the vaccine elicits aprotective immunity comprising an IgA mucosal immune response and an IgGsystemic immune response by administering parenterally (preferablyintramuscularly) to the subject no more than a single dose of anantigenic or vaccine composition comprising one or more types ofNorovirus antigens and optionally at least one effective adjuvant. Theinventors have surprisingly found that parenteral administration of theNorovirus vaccine compositions described herein induces a robust IgAresponse in addition to a strong IgG response. Typically, strong IgAresponses are only observed when vaccines are administered through amucosal route of administration.

In certain embodiments, the administration of the vaccine elicits aprotective immunity comprising an increase in the level of IgANorovirus-specific antibody secreting cells in the blood as compared tothe level in a human not receiving the vaccine. In some embodiments, theadministration of the vaccine elicits a protective immunity comprisingan increase in the level of IgA Norovirus-specific antibody secretingcells in the blood as compared to the level in the human beforereceiving the vaccine. In one embodiment, the IgA Norovirus-specificantibody secreting cells are CCR10+, CD19+, CD27+, CD62L+, and α4β7+.Antibody secreting cells with this marker profile are capable of homingto both peripheral lymphoid tissue, such as Peyer's patch in the gut,and mucosal lymphoid tissue, such as the gut mucosa. In one embodiment,the number of CCR10+, CD19+, CD27+, CD62L+, and α4β7+ IgA antibodysecreting cells is greater than about 500, about 700, about 1,000, about1,500, or greater than about 2,000 cells per 1×10⁶ peripheral bloodmonocytes. In another embodiment, the IgA Norovirus-specific antibodysecreting cells are CCR10+, CD19+, CD27+, CD62L−, and α4β7+. Antibodysecreting cells with this marker profile generally exhibit homing onlyto mucosal sites and can be indicative of a memory B-cell response. Insome embodiments in which the vaccine is administered intramuscularly,the number of CCR10+, CD19+, CD27+, CD62L−, and α4β7+ IgA antibodysecreting cells is greater than about 5,000, about 6,500, about 7,000,about 10,000, about 13,000, about 15,000, or greater than about 20,000cells per 1×10⁶ peripheral blood monocytes.

Similar findings have been observed with vaccines for other viruses,such as rotavirus. For rotavirus vaccines, there is controversy overwhether serum antibodies are directly involved in protection or merelyreflect recent infection (Jiang, 2002; Franco, 2006). Defining suchcorrelates of protection is particularly difficult in the context ofdiarrheal diseases such as rotavirus or norovirus, where preclinicalstudies inferring protection may be multifaceted with contributions frommucosal immunity (such as intestinal IgA), cytokine elaboration, andcell mediated immunity. The difficulty in measuring such immuneresponses during clinical development, and the lack of correlation toserum antibody measurements, requires that the effectiveness of avaccine for these types of viruses can only be demonstrated throughhuman clinical challenge experiments.

As mentioned above, administration of a vaccine composition of thepresent invention prevents and/or reduces at least one symptom ofNorovirus infection. Symptoms of Norovirus infection are well known inthe art and include nausea, vomiting, diarrhea, and stomach cramping.Additionally, a patient with a Norovirus infection may have a low-gradefever, headache, chills, muscle aches, and fatigue. The invention alsoencompasses a method of inducing a protective immune response in asubject experiencing a Norovirus infection by administering to thesubject a vaccine formulation of the invention such that at least onesymptom associated with the Norovirus infection is alleviated and/orreduced. A reduction in a symptom may be determined subjectively orobjectively, e.g., self assessment by a subject, by a clinician'sassessment or by conducting an appropriate assay or measurement (e.g.body temperature), including, e.g., a quality of life assessment, aslowed progression of a Norovirus infection or additional symptoms, areduced severity of Norovirus symptoms or suitable assays (e.g. antibodytiter, RT-PCR antigen detection, and/or B-cell or T-cell activationassay). An effective response may also be determined by directlymeasuring (e.g., RT-PCR) virus load in stool samples, which reflects theamount of virus shed from the intestines). The objective assessmentcomprises both animal and human assessments.

The invention also provides a method of generating antibodies to one ormore Norovirus antigens, said method comprising administration of avaccine composition of the invention as described above to a subject.These antibodies can be isolated and purified by routine methods in theart. The isolated antibodies specific for Norovirus antigens can be usedin the development of diagnostic immunological assays. These assayscould be employed to detect a Norovirus in clinical samples and identifythe particular virus causing the infection (e.g. Norwalk, Houston, SnowMountain, etc.). Alternatively, the isolated antibodies can beadministered to subjects susceptible to Norovirus infection to conferpassive or short-term immunity.

The invention will now be illustrated in greater detail by reference tothe specific embodiments described in the following examples. Theexamples are intended to be purely illustrative of the invention and arenot intended to limit its scope in any way.

EXAMPLES Example 1. Dose Escalation, Safety and Immunogenicity Study ofIntramuscular Norovirus Bivalent Virus-Like-Particle (VLP) Vaccine inHumans (LV03-104 Study), Cohort A

This example describes Cohort A of a randomized, multi-site,dose-escalation study of the safety and immunogenicity of four dosagelevels of an intramuscular (IM) Norovirus Bivalent VLP Vaccineadjuvanted with monophosphoryl lipid A (MPL) and aluminum hydroxide(AlOH) compared to placebo in adult subjects. Approximately 48 subjects18 to 49 years of age were enrolled in the cohort. Subjects received twodoses of the vaccine or placebo, by intramuscular (IM) injection, 28days apart using a 1.5 inch (38 mm) needle.

The Norovirus Bivalent VLP Vaccine contained genogroup I, genotype 1(GI.1) and genogroup II, genotype IV (GII.4) VLPs as the antigens, andMonophosphoryl Lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants,sodium chloride (NaCl) and L-histidine (L-His) as buffer (pH 6.3-6.7),ethanol and water for injection. The composition of the intramuscularNorovirus Bivalent VLP Vaccine is summarized in Table 1. The GII.4 VLPscomprised a capsid sequence of SEQ ID NO: 1, which was derived fromthree GII.4 strains.

TABLE 1 Final Drug Product Composition for Four IM Norovirus BivalentVLP Vaccine Formulations per 0.5 mL Formulation GI.1- GII.4 VLP VLP MPLAl* NaCl L-His Ethanol (μg) (μg) (μg) (mg) (mg) (mg) (mg) 10 μg 5 5 500.5 4.38 1.55 19.7 Dosage 30 μg 15 15 50 0.5 4.38 1.55 19.7 Dosage 100μg 50 50 50 0.5 4.38 1.55 19.7 Dosage 300 μg 150 150 50 0.5 4.38 1.5519.7 Dosage *as Aluminum Hydroxide

Placebo was sterile normal saline for injection (0.9% NaCl andpreservative-free). The dose escalation of the vaccine was conducted asfollows: after appropriate screening for good health, subjects in CohortA were enrolled sequentially into each of four dosage groups of ˜12subjects each (Dosage Groups A1, A2, A3, and A4). Dosage Groups A1, A2,A3, and A4 represent bivalent antigenic dosages of 5/5 μg, 15/15 μg,50/50 μg, and 150/150 μg, respectively, of the G I.1 and GII.4norovirus. Subjects in each dosage group were randomized 5:1 to receivevaccine or placebo. Subjects in Dosage Group A1 received theirrespective randomized treatment (10 subjects received 5/5 μg vaccine and2 subjects received placebo). Subjects were followed for safetyassessment by review of the symptoms recorded on the memory aid (Days0-7) and interim medical histories from the Day 7, 21, 28, 35, and 56visits. Safety data was reviewed by the Central Safety Monitor (CSM).After the 7-day post Dose 2 safety data (Study Day 35) were availablefor review from subjects in Dosage Group A1 and considered acceptable,subjects in Dosage Group A2 were eligible to receive their initial dose.The same rule applied for dosing in the subsequent dosage groups; thatis, after the 7-day post Dose 2 safety data (Study Day 35) wereavailable for review from a dosage group, the next dosage group waseligible to receive their initial dose.

At the end of enrollment in Cohort A, approximately 10 subjects in eachDosage Group received vaccine (total of 40 vaccinees) and 2 subjects ineach group received saline (total of approximately 8 saline controlrecipients).

The subjects kept a daily memory aid of solicited symptoms includingfour local injection site reactions, such as pain, tenderness, redness,and swelling, and 10 systemic signs or symptoms including daily oraltemperature, headache, fatigue, muscle aches, chills, joint aches andgastrointestinal symptoms of nausea, vomiting, diarrhea, abdominalcramps/pain for Days 0 through 7 after each dose of IM NorovirusBivalent VLP Vaccine or control. The redness and swelling at theinjection site was measured and recorded daily for 7 days after eachinjection.

Interim medical histories were obtained at each follow-up visit on Days7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 and at the follow-uptelephone call on Day 265+14; subjects were queried about interimillness, doctor's visits, any serious adverse events (SAEs), and onsetof any significant new medical conditions. Subjects had a CBC with WBCdifferential and platelet count, and serum BUN, creatinine, glucose,AST, and ALT assessed at screening and on Days 21 and 35 (˜7 days aftereach dose) to assess continuing eligibility and safety, respectively.

Blood from subjects was collected before vaccination on Day 0 and onDays 7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 to measure serumantibodies (IgG, IgA, and IgM separately and combined) to IM NorovirusBivalent VLP Vaccine by enzyme-linked immunosorbent assays (ELISA).Serum carbohydrate blocking activity and serum HAI antibodies were alsomeasured. For subjects in Cohort A, antibody secreting cells (ASCs),homing markers, memory B cells and cellular immune responses wereassayed.

The following methods were used to analyze the blood samples collectedfrom immunized individuals or individuals receiving the placebo.

Serum Antibody Measurements by ELISA

Measurement of antibodies to norovirus by ELISA was performed for allsubjects, using purified recombinant Norovirus VLPs (GI.1 and GII.4separately) as target antigens to screen the coded specimens. Briefly,norovirus VLPs in carbonate coating buffer pH 9.6 were used to coatmicrotiter plates. Coated plates were washed, blocked, and incubatedwith serial two-fold dilutions of test serum followed by washing andincubation with enzyme-conjugated secondary antibody reagents specificfor human total IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM. Appropriatesubstrate solutions were added, color developed, plates read and theIgG, IgA and IgM endpoint titers determined in comparison to a referencestandard curve for each antibody class. Geometric mean titers (GMTs),geometric mean fold rises (GMFRs) and seroresponse rates for each groupwas determined. Seroresponse was defined as a 4-fold increase inantibody titer compared to pre-immunization titers.

Norovirus Carbohydrate Histo-Blood-Group Antigens (HBGA) BlockingActivity

Blocking assays to measure the ability of serum antibodies to inhibit NVVLP binding to H type 1 or H type 3 synthetic carbohydrates wereperformed as previously described (Reeck et al. (2010) J Infect Dis,Vol. 202(8):1212-1218). Briefly, NV VLPs for the blocking assays wereincubated with an equal volume of serum, and serially two-fold dilutedfrom a starting dilution of 1:25. Neutravidin-coated, 96-well microtiterplates were washed and coated with 2.5 μg/mL of either syntheticpolyvalent H type 1-PAA-biotin or polyvalent H type 3-PAA-biotin. Thesera-VLP solutions were added. Plates were washed and rabbit polyclonalsera specific to NV VLPs was added, washed, and followed by incubationwith horseradish peroxidase conjugated goat anti-rabbit IgG. The colorwas developed with tetramethylbenzidine peroxidase liquid substrate andstopped with 1M phosphoric acid. Optical density was measured at 450.Positive and negative controls were performed. Fifty-percent blockingtiters (BT50) were determined, defined as the titer at which OD readings(after subtraction of the blank) are 50% of the positive control. Avalue of 12.5 was assigned to samples with a BT50 less than 25.Geometric mean titers (GMTs), geometric mean fold rises (GMFRs) andseroresponse rates for each group were determined. Seroresponse wasdefined as a 4-fold increase in antibody titer compared topre-immunization titers. A blocking control serum sample was used as aninternal control. An assay to confirm the specificity of the blockingwas performed using the same protocol for the blocking assay with thefollowing exceptions: after coating with carbohydrate, sera wasincubated directly on the plate without first pre-incubating with VLP.After washing, VLPs were incubated on the plate and detected as for theblocking assay.

Norovirus Hemagglutination Antibody Inhibition (HAI) Assay

Vaccine-induced antibodies were examined for the capacity to inhibithemagglutination of 0-type human RBCs by the norovirus VLPs aspreviously described (El Kamary et al. (2010) J Infect Dis, Vol.202(11): 1649-58). HAI titers were calculated as the inverse of thehighest dilution that inhibited hemagglutination with a compact negativeRBC pattern and are presented as GMTs, GMFRs and ≥4-fold rises.

Norovirus GI.1 and GII.4 VLPs were separately serially diluted andincubated with an equal volume of a 0.5% human RBC suspension in a96-well V bottom plate. The amount of norovirus VLP antigenscorresponding to 4 HA units were determined and confirmed by backtitration. Test sera were heat inactivated at 56 C for 30 minutes andtreated with freshly prepared 25% Kaolin suspension. To eliminate seruminhibitors, test samples were pre-adsorbed with RBCs. The HAI assay wereperformed as follows: pre-treated sera (diluted 2-fold in PBS pH 5.5)were added to 96 well V-plates and incubated with an equal volume ofNorovirus GI.1 and GII.4 VLP antigen, respectively, containing 4 HAunits. A suspension of 0.5% RBCs was added to each well and platesincubated for an additional 90 minutes at 4 C. Wells containing only PBSor antigen without serum served as negative and positive controls,respectively. Geometric mean titers (GMTs), geometric mean fold rises(GMFRs) and seroresponse rates for each group were determined.Seroresponse was defined as a 4-fold increase in antibody titer comparedto pre-immunization titers.

Antibody Secreting Cell Assays

PBMCs were isolated from approximately 60 mL of anti-coagulated blood onDays 0, 7+3, 28+3, and 35+3 after administration of IM NorovirusBivalent VLP Vaccine or placebo. Approximately 25 mL of blood for freshPBMC assays and 35 mL of blood for cryopreservation of PBMCs wasobtained. ASC assays detect cells secreting antibodies to norovirus VLPs(Tacket et al. (2000) J. Infect. Dis., Vol. 182:302-305; Tacket et al.(2003) Clin. Immunol., Vol. 108:241-247; El Kamary et al. (2010) JInfect Dis, Vol. 202(11): 1649-58). Fresh PBMCs were evaluated for ASCfrequency and determination of homing markers from a subset of subjects.Cryopreserved PBMCs from subjects participating in Cohort A wereevaluated for ASC frequency. The response rate and mean number of ASCper 10⁶ PBMCs at each time point for each group are described. Apositive response is defined as a post-vaccination ASC count per 10⁶PBMCs that is at least 3 standard deviations (SD) above the meanpre-vaccination count for all subjects (in the log metric) and at least8 ASC spots, which corresponds to the mean of medium-stimulated negativecontrol wells (2 spots) plus 3 SD as determined in similar assays.

Measurement of Norovirus Virus-Specific Memory B-Cells

Anti-coagulated blood was collected only in Cohort A subjects(approximately 25 mL on Days 0, 28, 56 and 180) to measure memory Bcells on days 0, 28, 56 and 180 after vaccination using an ELISpot assaypreceded by in vitro antigen stimulation (Crotty et al. (2004) J.Immunol. Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol.Methods, Vol. 313:110-118). Peripheral blood mononuclear cells (5×10⁶cells/mL, 1 mL/well in 24-well plates) were incubated for 4 days withnorovirus GI.1 and GII.4 VLP antigens separately to allow for clonalexpansion of antigen-specific memory B cells and differentiation intoantibody secreting cells. Controls included cells incubated in the sameconditions in the absence of antigen and/or cells incubated with anunrelated antigen. Following stimulation, cells were washed, counted,and transferred to ELISpot plates coated with Norwalk VLP. To determinefrequency of virus-specific memory B cells per total Ig-secreting Blymphocytes, expanded B cells were also added to wells coated withanti-human IgG and anti-human IgA antibodies. Bound antibodies wererevealed with HRP-labeled anti-human IgG or anti-human IgA followed byappropriate substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2and IgG1-4) are also used to determine antigen-specific subclassresponses that may be related with distinct effector mechanisms andlocations of immune priming. Spots were counted with an ELISpot reader.The expanded cell populations for each subject were examined by flowcytometry to confirm their memory B cell phenotype, i.e. CD19+, CD27+,IgG+, IgM+, CD38+, IgD, among others (Crotty et al. (2004) J. Immunol.Methods, Vol. 286:111-122.; Li et al. (2006) J. Immunol. Methods, Vol.313:110-118).

Cellular Immune Responses

Anti-coagulated blood (approximately 25 mL on Days 0, 28, 56, and 180)from subjects in Cohort A were collected as coded specimens and thePBMCs isolated and cryopreserved in liquid nitrogen for possible futureevaluation of CMI responses to norovirus GI.1 and GII.4 VLP antigens.Assays that are performed include PBMC proliferative and cytokineresponses to norovirus GI.1 and GII.4 VLP antigens by measuringinterferon (IFN)-γ and interleukin (IL)-4 levels among others accordingto established techniques (Samandari et al. (2000) J. Immunol., Vol.164:2221-2232; Tacket et al. (2003) Clin. Immunol., Vol. 108:241-247). Tcell responses are also evaluated.

Results

Safety assessment included local and systemic solicited symptoms for 7days and unsolicited symptoms for 28 days after each dose. SeriousAdverse Events are monitored for 12 months. Immunogenicity was assessedwith serum obtained prior to and after each vaccination for Pan-ELISAantibodies (IgG, IgA and IgM combined) and peripheral blood mononuclearcells (PBMCs) for IgG and IgA antibody secreting cells (ASC) viaElispot.

All four dosage groups have been enrolled for Cohort A with post dosetwo safety data available from all four dosage groups (40 vaccineestotal). Among the 40 vaccinees, pain or tenderness were the most commonlocal symptoms reported after either dose, whereas swelling or rednesswas infrequent. No severe local symptoms were reported. Systemicsymptoms of headache, myalgia, or malaise after either dose werereported by less than half of the vaccinees. No vaccinees reportedfever. No related SAEs were reported.

As shown in FIGS. 1A-3B, robust anamnestic Pan-ELISA antibody responses(combined IgG, IgA, and IgM) were observed to both VLP antigens 7 daysafter the first dose of the lowest dosage (5 μg GI.1+5 μg of GII.4VLPs). The second dose did not boost the post dose one responses.Similar results were observed for antigen-specific serum IgG and serumIgA responses measured separately (FIGS. 4A-9B). Dose-dependentresponses were observed for the antibody responses to both antigens(FIGS. 1A-9B). However, the maximal response to the GI.1 VLP appeared tobe achieved with a lower dose than the maximal response to the GII.4 VLP(15 μg vs. 50 μg). Interestingly, the single dose of the intramuscularlyadministered Norovirus bivalent vaccine induced a surprisingly,significantly greater antigen-specific antibody titer than the titerinduced by two doses of an intranasally administered monovalent VLPvaccine comprising a 20 fold higher VLP dose (FIG. 10; compare LV03-1045 μg group to LV01-103 100 μg group). Moreover, the low dose (5 μg), IMbivalent Norovirus vaccine produced a Norovirus-specific antibody titersimilar to that induced in humans exposed to the native Norovirus (FIG.10).

Robust IgG and IgA Elispot responses were also observed at 7 days afterthe first dose of the lowest dosage (5 μg) for both VLP antigens (Table2). Notably, the antibody secreting cell (ASC) responses were biased toIgA vs. IgG and ASCs exhibited a mucosal homing (alpha 4/beta7) andchemokine (CCR10) receptor phenotype as assessed by flow cytometry(FIGS. 11A-11B; Table 3). As shown in Table 3, a greater number of ASCsexhibit mucosal homing markers (beta 7+, CD62L−) as compared to dualmucosal/peripheral homing markers (beta 7+, CD62L+). Table 4 shows thepercentage of memory B cells per 10⁶ peripheral blood monocytes thatrespond to the VLP antigens. A larger percentage of antigen-specificmemory B cells also express mucosal homing markers as compared to thedual mucosal/peripheral or peripheral homing markers. Similar responseswere also observed in recipients who received the 15 μg and 50 μg doses(Tables 2-4).

TABLE 2 Day 7, Characterization of PBMC response. Approximation ofAntibody Secreting Cells (ASCs)/million CD19+ cells. ASC/million CD19+cells - Day 7 Vaccine Response Percent Vaccine SpecificNorovirus-specific IgA IgG IgA IgG Specific to Specific to Percent ofTotal B cells GI.1 GI.1 GII.4 GII.4 GI.1 GII.4 Circulating PBMCGeometric mean A1 30947 13807 10947 3945 4.48% 1.49% 5.96% 5 μg dose (n= 5) Standard 6674 9780 3651 2261 Deviation A1 5 μg dose Geometric meanA2 25296 17004 7108 4336 4.23% 1.14% 5.37% 15 μg dose (n = 4) Standard10846 18770 6055 5697 Deviation A2 15 μg dose Geometric mean A3 3615820572 14103 2549 5.67% 1.67% 7.34% 50 μg dose (n = 4) Standard 11470 4187627 2230 Deviation A3 50 μg dose Geometric mean A4 34183 9566 2621311310 4.37% 3.75% 8.13% 150 μg dose (n = 4) Standard 32938 4466 8976915226 Deviation A4 150 μg dose Placebo (n = 2) 0 152 0 108 0.02% 0.01%0.03%

TABLE 3 ASC markers in vaccine and placebo recipients by flowcytometry-Day 7 Percent Total Vaccine Specific Vaccine Specific % ofTotal % of % Total % of % Total ASC Total per million cells* Percent ofTotal* CD19+ B CD27+, CD38+, CD27+, CD38+, CD27+, CD38+, CD27+, CD38+,Circulating cells that are CCR10+_Beta 7+, CCR10+ Beta 7+, CCR10+, Beta7+ CCR10+, Beta 7+ PBMC Mucosal CD27+ & CD38+ CD62L− CD62L+ CD62L(+)&(−)CD62L(+)&(−) Homing Geometric mean A1 25.10% 6.86% 1.06% 2.78% 16560.17% 5 μg dose (n = 5) Standard 10.45 3.13 1.01 Deviation A1 5 μg doseGeometric mean A2 12.99% 16.98% 2.43% 4.63% 1355 0.14% 15 μg dose (n =4) Standard 9.13 1.56 0.23 Deviation A2 15 μg dose Geometric mean A331.71% 26.43% 3.63% 12.01% 23915 2.39% 50 μg dose (n = 4) Standard 6.321.82 1.38 Deviation A3 50 μg dose Geometric mean A4 33.46% 30.06% 5.68%15.74% 31350 3.14% 150 μg dose (n = 4) Standard 9.86 2.97 1.70 DeviationA4 150 μg dose Placebo (n = 2) 1.26% 22.00% 0.87% 1.20% 5 0.001% *Assumes the majority of ASCs are norovirus-specific.

TABLE 4 Memory B cell responses in vaccine and placebo recipients-Day 7Percent Total Vaccine Specific % of Total % of % Total Memory Total permillion cells* Vaccine Specific CD19+ B % of % Total CD27+, CD38+,CD27+, CD38+, CD27+, CD38+, Percent of Total* cells that are CD27+,CD38+, CD138+, CD138+, CCR10+, CD138+, CCR10+, Circulating CD27+, CD38+,CD138+, CCR10+ CCR10+_Beta 7+, Beta 7+ Beta 7+ PBMC Mucosal CD138+ Beta7+, CD62L− CD62L+ CD62L(+)&(−) CD62L(+)&(−) Homing Geometric mean A1 N/DN/D N/D N/D N/D N/D 5 μg dose Geometric mean A2 1.54% 11.58% 1.92% 0.21%61 0.01% 15 μg dose (n = 4) Standard 1.54 3.94 0.94 Deviation A2 15 μgdose Geometric mean A3 3.31% 16.10% 4.60% 0.68% 1364 0.14% 50 μg dose (n= 4) Standard 1.11 2.16 0.97 Deviation A3 50 μg dose Geometric mean A41.56% 16.90% 8.10% 0.39% 778 0.08% 150 μg dose (n = 4) Standard 0.223.26 4.57 Deviation A4 150 μg dose Placebo (n = 1) 0.10% 12.50% 0.00%0.01% 0   0% *Assumes the majority of ASCs are norovirus-specific.

In the absence of an available direct viral neutralization assay due tothe inability to culture Norovirus in vitro, functional assays whichserve as substitutes for viral neutralization assays were conducted tomeasure functional antibodies in vaccinees.

Using the carbohydrate H antigen blocking activity assay describedabove, the inhibition of GI.1 VLP binding to H antigen mediated byvaccine-induced serum antibodies was measured. Data are presented asgeometric mean fold rise (GMFR) and seroresponse (4-fold rise) in Table5, and as geometric mean titer (GMT) in Table 6. Surprisingly, afterjust one intramuscular injection of the vaccine formulation, significantcarbohydrate blocking activity was observed in all dose groups; in fact,the administration of a second dose of vaccine did not significantlyincrease blocking activity compared to post-dose 1 levels. Theinhibition of binding activity was maintained throughout the testingperiod, up to 56 days post dose 1.

TABLE 5 Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GI.1Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise) Study Day28 Days Post Dose 1 7 Days Post Dose 2 28 Days Post Dose 2 7 Days PostDose 1 21 Days Post Dose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 DaysPost Dose 1) GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-FoldTreatment (95% Rise (95% Rise (95% Rise (95% Rise (95% Rise Group N CI)(95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) 5/5mcg 9 26.6 88.9 9 25.1 88.9 9 19.7 100.0 9 20 88.9 9 16.6 77.8 VLPVaccine (8.3, (51.8, (8.9, (51.8, (8.2, (66.4, (7.7, (51.8, (5.7, (40.0,85.1) 99.7) 70.3) 99.7) 47.1) 100.0) 51.7) 99.7) 48.1) 97.2) 15/15 mcg 833.2 100.0 8 25.5 100.0 8 18.5 100.0 7 22.2 100.0 7 8.4 57.1 VLP Vaccine(13.6, (63.1, (10.5, (63.1, (8.4, (63.1, (8.8, (59.0, (2.4, (18.4, 80.8)100.0) 61.8) 100.0) 40.6) 100.0) 56) 100.0) 29.6) 90.1) 50/50 mcg 1038.6 100.0 10 27.9 100.0 10 20.9 100.0 10 19 100.0 9 10.2 77.8 VLPVaccine (18.3, (69.2, (13.4, (69.2, (10, (69.2, (9.9, (69.2, (4.6,(40.0, 81.6) 100.0) 58) 100.0) 43.5) 100.0) 36.4) 100.0) 22.8) 97.2)150/150 mcg 7 30.6 100.0 8 19.4 100.0 8 16.3 100.0 8 18.8 100.0 8 23.8100.0 VLP Vaccine (16.3, (59.0, (13.1, (63.1, (11.7, (63.1, (12.8,(63.1, (17, (63.1, 57.6) 100.0) 28.5) 100.0) 22.6) 100.0) 27.5) 100.0)33.3) 100.0) Placebo 8 0.9 0.0 8 0.8 0.0 8 0.8 0.0 8 0.8 0.0 8 0.6 0.0(0.8, (0.0, (0.7, (0.0, (0.6, (0.0, (0.7, (0.0, (0.3, (0.0, 1) 36.9)1.1) 36.9) 1.1) 36.9) 1.1) 36.9) 1.2) 36.9) Results based on allsubjects receiving both doses of study product. Two subjects' datapoints are excluded due to a possible mix-up of specimens; one of thesedata points is a baseline specimen resulting in the subject not havingfold rise data available for any time point.

TABLE 6 Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GI.1Geometric Mean Titer (GMT) Study Day 28 Days Post Dose 1 7 Days PostDose 2 28 Days Post Dose 2 Pre-Dose 1 7 Days Post Dose 1 21 Days PostDose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 Days Post Dose 1)Treatment GMT GMT GMT GMT GMT GMT Group N (95% CI) N (95% CI) N (95% CI)N (95% CI) N (95% CI) N (95% CI) 5/5 mcg 9 28.9 9 768.5 9 723.7 9 568 9577.1 9 478.3 VLP Vaccine (12.7, (344.1, (398.1, (321.8, (351.2, (293.3,65.9) 1716) 1316) 1003) 948.3) 780.1) 15/15 mcg 8 24.9 8 826.1 8 634.3 8459.8 7 610.3 7 230.9 VLP Vaccine (12.7, (524.9, (285.9, (225.3, (354.6,(105.2, 48.7) 1300) 1407) 938.6) 1050) 506.7) 50/50 mcg 10 17.3 10 669.210 483.7 10 362.4 10 328.9 9 184 VLP Vaccine (9.9, (329.1, (258.7,(192.9, (191.9, (97.2, 30.3) 1361) 904.2) 680.7) 563.8) 348.3) 150/150mcg 8 15.5 7 435 8 300.7 8 252.7 8 291.5 8 369.7 VLP Vaccine (11.1,(262.5, (173.9, (146.7, (171.5, (233.8, 21.8) 720.8) 520) 435.2) 495.4)584.6) Placebo 8 29 9 24.6 9 22.5 9 22.2 8 24.6 8 18.3 (9.1, (9.8, (8.8,(8.9, (8.4, (10.1, 92.8) 62.1) 57.3) 55.5) 72.6) 33.3) Results based onall subjects receiving both doses of study product. Two subjects' datapoints are excluded due to a possible mix-up of specimens.

Similarly, carbohydrate blocking activity of serum antibodies againstGII.4 VLPs was measured. A significant response was observed in alldosing groups as measured by GMFR and seroresponse (Table 7) as well asGMT (Table 8). Similar to the antibody-mediated blocking of GI.1 bindingdescribed above, robust blocking of GII.4 VLP carbohydrate bindingactivity was detected after just one dose, and a second dose did notappear to enhance the blocking activity.

TABLE 7 Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GII.4Geometric Mean Fold Rise (GMFR) and Seroresponse (4-Fold Rise) Study Day28 Days Post Dose 1 7 Days Post Dose 2 28 Days Post Dose 2 7 Days PostDose 1 21 Days Post Dose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 DaysPost Dose 1) GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-FoldTreatment (95% Rise (95% Rise (95% Rise (95% Rise (95% Rise Group N CI)(95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) 5/5mcg 9 5 33.3 9 5.9 55.6 9 4.7 44.4 9 4.7 44.4 9 5 55.6 VLP Vaccine (1.6,(7.5, (1.7, (21.2, (1.4, (13.7, (1.6, (13.7, (1.6, (21.2, 16.1) 70.1)20.3) 86.3) 15.7) 78.8) 13.8) 78.8) 15.9) 86.3) 15/15 mcg 8 11 62.5 89.2 62.5 8 7.4 62.5 7 7 57.1 7 5.6 57.1 VLP Vaccine (2.7, (24.5, (3,(24.5, (2.5, (24.5, (2.1, (18.4, (2.3, (18.4, 45.3) 91.5) 27.9) 91.5)21.8) 91.5) 23) 90.1) 14) 90.1) 50/50 mcg 10 18.6 70.0 10 12.2 70.0 108.4 70.0 10 8.7 70.0 9 5.2 66.7 VLP Vaccine (4.9, (34.8, (3.8, (34.8,(2.9, (34.8, (2.9, (34.8, (2.2, (29.9, 70.8) 93.3) 39.4) 93.3) 24.1)93.3) 26.1) 93.3) 12) 92.5) 150/150 mcg 7 10.1 57.1 8 5.5 50.0 8 4.450.0 8 4.3 37.5 8 3.1 25.0 VLP Vaccine (2, (18.4, (1.9, (15.7, (1.6,(15.7, (1.7, (8.5, (1.3, (3.2, 51.8) 90.1) 16.5) 84.3) 12.2) 84.3) 10.7)75.5) 7.2) 65.1) Placebo 8 1 0.0 8 1.1 0.0 8 1.3 12.5 8 1.8 12.5 8 212.5 (0.9, (0.0, (1, (0.0, (0.9, (0.3, (0.5, (0.3, (0.7, (0.3, 1.1)36.9) 1.3) 36.9) 2.1) 52.7) 6.7) 52.7) 6.1) 52.7) Results based on allsubjects receiving both doses of study product. Two subjects' datapoints are excluded due to a possible mix-up of specimens; one of thesedata points is a baseline specimen resulting in the subject not havingfold rise data available for any time point.

TABLE 8 Carbohydrate Blocking Activity (HBGA BT50), Anti-Norovirus GII.4Geometric Mean Titer (GMT) Study Day 28 Days Post Dose 1 7 Days PostDose 2 28 Days Post Dose 2 Pre-Dose 1 7 Days Post Dose 1 21 Days PostDose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 Days Post Dose 1)Treatment GMT GMT GMT GMT GMT GMT Group N (95% CI) N (95% CI) N (95% CI)N (95% CI) N (95% CI) N (95% CI) 5/5 mcg 9 40.3 9 202.1 9 236.9 9 189.79 188.7 9 201.6 VLP Vaccine (18, (106.3, (133.4, (108.6, (118.7, (116.4,90) 384.3) 420.6) 331.3) 300.1) 349.5) 15/15 mcg 8 23.7 8 260.1 8 218.18 175.4 7 182.3 7 146.3 VLP Vaccine (12.8, (95.1, (104.2, (82.7, (89.1,(92.4, 43.8) 711.1) 456.3) 372) 372.8) 231.5) 50/50 mcg 10 28.4 10 527.210 345.2 10 238.3 10 246.5 9 160.2 VLP Vaccine (13.1, (271.1, (195.5,(139.4, (138.7, (107.4, 61.5) 1025) 609.6) 407.3) 438.2) 238.8) 150/150mcg 8 63 7 721.8 8 347.7 8 277 8 267.9 8 193.5 VLP Vaccine (24.8,(344.6, (186.1, (145.6, (158.7, (121.6, 160.4) 1512) 649.5) 527) 452.2)308.2) Placebo 8 24.1 9 22.8 9 24.9 9 29.1 8 44 8 48.2 (12.9, (12.6,(12.7, (15.1, (12.6, (16.7, 45) 41.6) 48.7) 56.1) 154.2) 139.5) Resultsbased on all subjects receiving both doses of study product. Twosubjects' data points are excluded due to a possible mix-up ofspecimens.

Hemagglutination Inhibition assays (HAI) were also utilized to test theresponse of serum antibodies from vaccinated subjects against targetNorovirus VLP antigens. Similar to carbohydrate H antigen bindingstudies, just one dose of VLP vaccine induced antibodies that inhibitedhemagglutination in all dosing groups, as measured by GMFR (Table 9),4-fold rise (Table 9), and GMT (Table 10). Though the level ofinhibition of hemagglutination was maintained through the last daytested (28 days post dose 2, 56 days post dose 1), the second dose ofVLP vaccine did not appear to enhance vaccine-induced antibody-mediatedinhibition of hemagglutination.

TABLE 9 Hemagglutination Inhibition Assay Anti-Norovirus GI.1 GeometricMean Fold Rise (GMFR) and Seroresponse (4-Fold Rise) Study Day 28 DaysPost Dose 1 7 Days Post Dose 2 28 Days Post Dose 2 7 Days Post Dose 1 21Days Post Dose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 Days PostDose 1) GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold GMFR 4-FoldTreatment (95% Rise (95% Rise (95% Rise (95% Rise (95% Rise Group N CI)(95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) 5/5mcg 9 5.4 77.8 9 7 88.9 9 6.1 88.9 9 6 77.8 9 6.3 88.9 VLP Vaccine (3,(40.0, (4.6, (51.8, (4.1, (51.8, (3.7, (40.0, (3.9, (51.8, 9.8) 97.2)10.7) 99.7) 9.3) 99.7) 9.5) 97.2) 10.3) 99.7) 15/15 mcg 8 8.9 100.0 89.5 87.5 8 7.1 75.0 7 8.5 85.7 7 8.1 100.0 VLP Vaccine (4.4, (63.1, (4,(47.3, (3.1, (34.9, (4.1, (42.1, (4.4, (59.0, 18) 100.0) 22.5) 99.7)16.1) 96.8) 17.7) 99.6) 15.2) 100.0) 50/50 mcg 10 22.4 100.0 10 16.7100.0 10 13.9 100.0 10 14.5 100.0 9 11.8 100.0 VLP Vaccine (11.6, (69.2,(9.3, (69.2, (8.1, (69.2, (9.3, (69.2, (6.3, (66.4, 43) 100.0) 29.8)100.0) 24) 100.0) 22.7) 100.0) 21.9) 100.0) 150/150 mcg 7 12.6 85.7 811.1 100.0 8 8.4 100.0 8 8.4 100.0 8 7.3 100.0 VLP Vaccine (5.7, (42.1,(6.4, (63.1, (5, (63.1, (5, (63.1, (4.5, (63.1, 28) 99.6) 19.3) 100.0)14) 100.0) 14) 100.0) 11.9) 100.0) Placebo 8 1 0.0 8 1 0.0 8 1 0.0 8 0.90.0 8 1 0.0 (0.8, (0.0, (0.9, (0.0, (0.9, (0.0, (0.7, (0.0, (0.8, (0.0,1.2) 36.9) 1.2) 36.9) 1.1) 36.9) 1.1) 36.9) 1.2) 36.9) Results based onall subjects receiving both doses of study product. Two subjects' datapoints are excluded due to a possible mix-up of specimens; one of thesedata points is a baseline specimen resulting in the subject not havingfold rise data available for any time point.

TABLE 10 Hemagglutination Inhibition Assay Anti-Norovirus GI.1 GeometricMean Titer (GMT) Study Day 28 Days Post Dose 1 7 Days Post Dose 2 28Days Post Dose 2 Pre-Dose 1 7 Days Post Dose 1 21 Days Post Dose 1(Pre-Dose 2) (35 Days Post Dose 1) (56 Days Post Dose 1) Treatment GMTGMT GMT GMT GMT GMT Group N (95% CI) N (95% CI) N (95% CI) N (95% CI) N(95% CI) N (95% CI) 5/5 mcg 9 26.4 9 143.5 9 185.3 9 162.1 9 157 9 167.4VLP Vaccine (14.1, (53.6, (88.6, (83.3, (80.3, (88.4, 49.4) 383.8)387.6) 315.6) 307.1) 316.8) 15/15 mcg 8 11.9 8 105.3 8 113.1 8 84.2 7103.3 7 99.2 VLP Vaccine (6.1, (56.1, (42.5, (31.1, (43.3, (46.9, 23.4)197.6) 301.3) 227.6) 246.6) 209.7) 50/50 mcg 10 7.2 10 160 10 119.2 1099.7 10 103.8 9 81.1 VLP Vaccine (5.3, (79.4, (60.4, (51.8, (58.4,(38.8, 9.6) 322.6) 235.1) 191.8) 184.5) 169.5) 150/150 mcg 8 9.2 7 114.18 101.6 8 77.2 8 77.2 8 67.3 VLP Vaccine (7.5, (50.9, (62, (51.8, (51.8,(44.7, 11.3) 255.7) 166.4) 115) 115) 101.3) Placebo 8 16.8 9 16.6 9 16.19 16.6 8 15.4 8 16.2 (10.1, (11.7, (10.7, (11, (10, (10.8, 28.1) 23.7)24.1) 25) 23.7) 24.4) Results based on all subjects receiving both dosesof study product. Two subjects' data points are excluded due to apossible mix-up of specimens.

Inhibition of hemagglutination was also achieved when the target VLP wasa mismatched virus. Vaccine-induced serum antibodies inhibitedhemagglutination by a Houston virus strain VLP, as measured by GMFR andseroresponse (Table 11) as well as GMT (Table 12). In this case, thehigher VLP vaccine doses afforded stronger responses, particularly asmeasured by 4-fold rise or GMT. GMFR and 4-fold rise were alsosignificantly increased when the target VLP was the 2003 Cincinnativirus strain, as measured just 7 days post dose 1 (Table 13).

TABLE 11 Hemagglutination Inhibition Assay (Houston Virus Strain VLP),Anti-Norovirus GII.4 Geometric Mean Fold Rise (GMFR) and Seroresponse(4-Fold Rise) Study Day 28 Days Post Dose 1 7 Days Post Dose 2 28 DaysPost Dose 2 7 Days Post Dose 1 21 Days Post Dose 1 (Pre-Dose 2) (35 DaysPost Dose 1) (56 Days Post Dose1) GMFR 4-Fold GMFR 4-Fold GMFR 4-Fold4-Fold GMFR 4-Fold Treatment (95% Rise (95% Rise (95% Rise GMFR Rise(95% Rise Group N CI) (95% CI) N CI) (95% CI) N CI) (95% CI) N (95% CI)(95% CI) N CI) (95% CI) 5/5 mcg 9 1.2 0.0 9 1.3 0.0 9 1.3 0.0 9 1.3 0.09 1.3 0.0 VLP Vaccine (1, (0.0, (0.9, (0.0, (0.9, (0.0, (1, (0.0, (1,(0.0, 1.5) 33.6) 1.7) 33.6) 1.7) 33.6) 1.7) 33.6) 1.6) 33.6) 15/15 mcg 81.7 12.5 8 1.6 12.5 8 1.5 0.0 7 1.5 0.0 7 1.6 0.0 VLP Vaccine (0.9,(0.3, (1.1, (0.3, (1.1, (0.0, (1, (0.0, (1.1, (0.0, 3) 52.7) 2.4) 52.7)2.2) 36.9) 2.2) 41.0) 2.4) 41.0) 50/50 mcg 10 2 10.0 10 1.6 10.0 10 1.710.0 10 1.5 10.0 9 1.3 11.1 VLP Vaccine (0.9, (0.3, (0.8, (0.3, (0.9,(0.3, (0.9, (0.3, (0.8, (0.3, 4.3) 44.5) 3.1) 44.5) 3.1) 44.5) 2.4)44.5) 2) 48.2) 150/150 mcg 7 4.3 57.1 8 2.6 50.0 8 1.9 12.5 8 1.9 12.5 81.7 12.5 VLP Vaccine (1.5, (18.4, (1.2, (15.7, (1.1, (0.3, (1.1, (0.3,(1.1, (0.3, 12.4) 90.1) 5.8) 84.3) 3.3) 52.7) 3.5) 52.7) 2.6) 52.7)Placebo 8 1 0.0 8 1.1 0.0 8 1.1 0.0 8 1.2 12.5 8 1.1 12.5 (0.9, (0.0,(0.9, (0.0, (1, (0.0, (0.8, (0.3, (0.7, (0.3, 1.1) 36.9) 1.3) 36.9) 1.2)36.9) 1.9) 52.7) 1.8) 52.7) Results based on all subjects receiving bothdoses of study product. Two subjects' data points are excluded due to apossible mix-up of specimens; one of these data points is a baselinespecimen resulting in the subject not having fold rise data availablefor any time point.

TABLE 12 Hemagglutination Inhibition Assay (Houston Virus Strain VLP),Anti-Norovirus GII.4 Geometric Mean Titer (GMT) Study Day 28 Days PostDose 1 7 Days Post Dose 2 28 Days Post Dose 2 Pre-Dose 1 7 Days PostDose 1 21 Days Post Dose 1 (Pre-Dose 2) (35 Days Post Dose 1) (56 DaysPost Dose 1) Treatment GMT GMT GMT GMT GMT GMT Group N (95% CI) N (95%CI) N (95% CI) N (95% CI) N (95% CI) N (95% CI) 5/5 mcg 9 143.5 9 177.49 180.8 9 180.8 9 189.1 9 180.8 VLP Vaccine (97.9, (122.7, (114.4,(114.4, (119.2, (122.7, 210.3) 256.5) 285.6) 285.6) 299.9) 266.3) 15/15mcg 8 129.8 8 218.3 8 207.5 8 200.2 7 169.5 7 187.2 VLP Vaccine (86.4,(129.2, (134.8, (132.8, (114.1, (119.1, 194.9) 368.8) 319.3) 301.7) 252)294.2) 50/50 mcg 10 161.9 10 323.8 10 252.5 10 273.9 10 242.5 9 195.2VLP Vaccine (119.7, (156.5, (130.4, (150.9, (150.2, (125, 219) 669.8)489.2) 497.2) 391.5) 304.9) 150/150 mcg 8 210.6 7 853.5 8 546.2 8 406.38 406.3 8 354.1 VLP Vaccine (108.8, (297.4, (237.6, (201.5, (180.3,(184.6, 407.5) 2449) 1255) 819.1) 915.4) 679.5) Placebo 8 148.9 9 150.19 157 9 167.4 8 183.6 8 162.4 (73.8, (80.1, (77.9, (91.4, (95, (88.5,300.3) 281.1) 316.5) 306.4) 354.5) 297.9) Results based on all subjectsreceiving both doses of study product. Two subjects' data points areexcluded due to a possible mix-up of specimens.

TABLE 13 Inhibition of Hemagglutination in placebo versus 50/50 μg VLPvaccine Hemagglutination Inhibition Assay (2003 Cincinnati Virus StrainVLP), Anti-Norovirus GII.4 Geometric Mean Fold Rise (GMFR) and GeometricSeroresponse (4-Fold Rise) Results by Treatment Group 7 Days Post Dose 1Treatment Group N GMFR (95% CI) 4-Fold Rise (95% CI) Placebo 2 1.0 (0.6,1.8)  0.0 (0.0, 84.2) 50/50 μg VLP Vaccine 10 4.4 (1.6, 11.9) 50.0(18.7, 81.3)

The results from this study demonstrated that the Bivalent IM NorovirusVLP vaccine was generally well tolerated. The immunogenicity datasuggested that a single vaccine dose may be sufficient to protectseropositive human adults. The results from the carbohydrate blockingactivity and hemagglutination inhibition assays provided furtherevidence that a single vaccine dose induced serum antibodies with potentanti-Norovirus activity. The magnitude and rapidity of the observedimmune responses following a single parenteral dose in humans weredramatic when compared to earlier immune responses reported by multiplenasal VLP vaccine administrations at much higher VLP dosages (El Kamaryet al. (2010) J Infect Dis, Vol. 202(11): 1649-1658). These responseswere also superior to those induced by orally administered NorovirusVLPs (Tacket et al. (2003) Clin Immunol 108:241-247; Ball et al. (1999,Gastroenterology 117:40-48) as well as those induced by Norovirus VLPsproduced by transgenic plants (Tacket et al. (2000) J Infect Dis182:302-305). In particular, this intramuscular vaccine formulationproduced anamnestic responses within seven days of immunization andmaximal serum antibody responses were observed after a single dose,including a significant IgA response and functional carbohydrateblocking activity and hemagglutination inhibition activity. Thus, thisNorovirus bivalent vaccine induced a strong, protective immune responsein humans that was superior to immune responses induced by any currentlyavailable Norovirus vaccine.

Example 2. Dose Escalation, Safety and Immunogenicity Study ofIntramuscular Norovirus Bivalent Virus-Like-Particle (VLP) Vaccine inHumans (LV03-104 Study)

The following example provides the remaining planned portion of theclinical study described in Example 1, wherein a randomized, multi-site,dose-escalation study is conducted in adults ≥18 years of age of thesafety and immunogenicity of four dosage levels of an intramuscular (IM)Norovirus Bivalent VLP Vaccine adjuvanted with monophosphoryl lipid A(MPL) and aluminum hydroxide (AlOH), compared to placebo. Subjects willreceive two doses of the vaccine or placebo, by intramuscular (IM)injection, 28 days apart using a 1.5 inch (38 mm) needle. This exampleis intended to further illustrate the principles of the presentinvention.

Cohort A has completed enrollment in the study and was described abovein Example 1. Cohort B contains ˜20 subjects 50-64 years of age. CohortC contains ˜30 subjects 65-85 years of age. Approximately 98 subjectsare enrolled in the study as a whole.

In Cohort B, ˜20 subjects 50-64 years of age are enrolled and randomized1:1 to receive vaccine (N=10) or placebo (N=10). After the 7-day postDose 2 safety data (Study Day 35) are available for review from subjectsin Cohort B, subjects in Cohort C are eligible to receive their initialdose. In Cohort C, ˜30 subjects 65 to 85 years of age are enrolled andrandomized 1:1:1 to receive vaccine adjuvanted with MPL and AlOH (N=10),or vaccine adjuvanted with AlOH alone, i.e. no MPL (N=10), or placebo(N=10). The antigen concentrations of the norovirus VLPs and of the AlOHin the two vaccine formulations to be evaluated in Cohort C areidentical; only the presence or absence of MPL is different.

The Norovirus Bivalent VLP Vaccine contains genogroup I, genotype 1(GI.1) and genogroup II, genotype IV (GII.4) VLPs as the antigens, andMonophosphoryl Lipid A (MPL) and aluminum hydroxide (AlOH) as adjuvants,sodium chloride (NaCl) and L-histidine (L-His) as buffer (pH 6.3-6.7),ethanol and water for injection. The GII.4 VLPs comprised a capsidsequence of SEQ ID NO: 1, which was derived from three GII.4 strains.

The single dosage of vaccine selected for further evaluation in CohortsB and C is the lowest dosage in Cohort A that results in the most robustand reproducible immune response that is also generally well tolerated.The Day 56 safety and immunogenicity data from subjects in Cohort A isreviewed by the CSM/SMC and the bivalent dosage is selected forevaluation in Cohorts B and C.

The subjects keep a daily memory aid of solicited symptoms includingfour local injection site reactions, such as pain, tenderness, redness,and swelling, and 10 systemic signs or symptoms including daily oraltemperature, headache, fatigue, muscle aches, chills, joint aches andgastrointestinal symptoms of nausea, vomiting, diarrhea, abdominalcramps/pain for Days 0 through 7 after each dose of IM NorovirusBivalent VLP Vaccine or control. The redness and swelling at theinjection site are measured and recorded daily for 7 days after eachinjection.

Interim medical histories are obtained at each follow-up visit on Days7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 and at the follow-uptelephone call on Day 265+14; subjects are queried about interimillness, doctor's visits, any serious adverse events (SAEs), and onsetof any significant new medical conditions. Subjects have a CBC with WBCdifferential and platelet count, and serum BUN, creatinine, glucose,AST, and ALT assessed at screening and on Days 21 and 35 (˜7 days aftereach dose) to assess continuing eligibility and safety, respectively.

Blood from subjects is collected before vaccination on Day 0 and on Days7+3, 21+3, 28+3, 35+3, 56+7, 180+14, and 393+14 to measure serumantibodies (IgG, IgA, and IgM separately and combined) to IM NorovirusBivalent VLP Vaccine by enzyme-linked immunosorbent assays (ELISA).Serum carbohydrate blocking activity and serum HAI antibodies are alsomeasured.

The methods described above for Cohort A are used to analyze the bloodsamples collected from immunized individuals or individuals receivingthe placebo. The results of the study will be employed in thedevelopment of a clinical protocol for administration of the vaccineformulations of the invention.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein, will become apparent to those skilled in the art from theforegoing description and accompanying drawings using no more thanroutine experimentation. Such modifications and equivalents are intendedto fall within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

The invention claimed is:
 1. A method of eliciting protective immunityagainst Norovirus in a human comprising administering parenterally tothe human no more than a single dose of a vaccine composition, saidcomposition comprising (i) genogroup I Norovirus virus-like particles(VLPs), wherein said genogroup I Norovirus VLPs comprise a capsidprotein derived from a genogroup I viral strain, and (ii) genogroup IINorovirus VLPs, wherein said genogroup II Norovirus VLPs comprise acapsid protein derived from a genogroup II viral strain, wherein saidgenogroup I Norovirus VLPs and genogroup II Norovirus VLPs are presentin the composition in different amounts, wherein said compositioncomprises about 15 μg to 50 μg of genogroup I Norovirus VLPs and about50 μg to 150 μg of genogroup II Norovirus VLPs, wherein said compositioninduces at least a three-fold increase in Norovirus-specific serumantibody titer as compared to the titer in the human prior toadministration of the composition.
 2. The method of claim 1, wherein thecomposition comprises about 15 μg of said genogroup I Norovirus VLPs. 3.The method of claim 1, wherein the composition comprises about 50 μg ofsaid genogroup II Norovirus VLPs.
 4. The method of claim 1, wherein thecomposition comprises about 15 μg of said genogroup I Norovirus VLPs andabout 50 μg of said genogroup II Norovirus VLPs.
 5. The method of claim1, wherein the composition induces at least a six-fold increase inNorovirus-specific serum antibody titer as compared to the titer in thehuman prior to administration of the composition.
 6. The method of claim1, wherein said Norovirus VLPs are monovalent VLPs or multivalent VLPs.7. The method of claim 1, wherein said genogroup I Norovirus VLPs areNorwalk virus VLPs and said genogroup II Norovirus VLPs are VLPsgenerated from expression of a consensus sequence of genogroup IINorovirus.
 8. The method of claim 1, wherein the composition furthercomprises at least one adjuvant.
 9. The method of claim 8, wherein saidat least one adjuvant is a toll-like receptor agonist.
 10. The method ofclaim 8, wherein the adjuvant is selected from monophosphoryl lipid Aand aluminum hydroxide.
 11. The method of claim 1, wherein saidcomposition further comprises a buffer.
 12. The method of claim 11,wherein said buffer is selected from the group consisting ofL-histidine, imidazole, succinic acid, tris, and citric acid.
 13. Themethod of claim 1, wherein the vaccine composition is administered tothe human by an intravenous, subcutaneous, intradermal, or intramuscularroute of administration.
 14. The method of claim 13, wherein the vaccinecomposition is administered to the human by an intramuscular route ofadministration.
 15. The method of claim 1, wherein the vaccinecomposition is formulated as a liquid.
 16. The method of claim 1,wherein the increase in Norovirus-specific antibody titer is inducedwithin seven days of administration of the single dose of thecomposition.
 17. The method of claim 1, wherein said vaccine compositionconfers protection from one or more symptoms of Norovirus infection. 18.The method of claim 8, wherein said at least one adjuvant is an aluminumhydroxide.